CA2456050A1 - Caax prenyl protease nucleic acids and polypeptides and methods of use thereof - Google Patents

Abstract

The present invention provides novel isolated prenyl protease polynucleotides and polypeptides encoded by the prenyl protease polynucleotides. Also provided are the antibodies that immunospecifically bind to a prenyl protease polypeptide or any derivative, variant, mutant or fragment of the prenyl protease polypeptide, polynucleotide or antibody. The invention additionally provides methods of constructing transgenic plants that have altered levels of prenyl protease polynucleotides and polypeptides. Methods for identifying prenyl protease enzymes substrates and inhibitors are also provided.

Description

CaaX Prenyl Protease Nucleic Acids and Polypeptides and Methods of Use Thereof FIELD OF THE INVENTION
The invention relates to novel plant CaaX prenyl protease polynucleotides and polypeptides. Also included are transgenic plants expressing the novel polynucleotides and polypeptides. Also included are transgenic plant cells, tissues and plants having novel phenotypes resulting from the expression of these polynucleotides in either the sense or antisense orientation.
BACKGROUND OF THE INVENTION
Most higher plants encounter at least transient decreases in relative water content at some stage of their life cycle and, as a result, have evolved a number of desiccation protection mechanisms. If however, the change in water deficit is prolonged the effects on the plants growth and development can be profound. Decreased water content due to drought, cold or salt stress can irreparably damage plant cells which in turn limits plant growth and crop productivity in agriculture.
Plants respond to adverse conditions of drought, salinity and cold with a variety of morphological and physiological changes. Although our understanding of plant tolerance mechanisms to these stresses is incomplete, the plant hormone abscisic acid 2o (ABA) is believed to be an essential mediator between environmental stimulus and plant responses. ABA levels increase in response to water deficits and exogenously applied ABA mimics many of the responses induced by water-stress. Once ABA is synthesized it causes the closure of the leaf stomata thereby decreasing water loss through transpiration.
The identification of genes that transduce ABA into a cellular response opens the possibility of exploiting these regulators to enhance desiccation tolerance in crop species.
In principle, these ABA signaling genes can be coupled with the appropriate controlling elements to allow optimal plant growth, development and productivity. Thus, not only would these genes allow the genetic tailoring of crops to withstand transitory 3o environmental stresses, but they should also broaden the environments where traditional crops can be grown.

The recent isolation of an Arabidopsis mutant, eral, is hypersensitive to ABA
and has been shown to also be tolerant to conditions of water deprivation.
ERAI has been identified as a (3 subunit of farnesyl transferase knockout mutant in.
Farnesyl transferase is a heterodimeric enzyme that provides the specific addition of a farnesyl pyrophosphate moiety onto the substrate target sequence. The target sequence is defined as a sequence of four amino acids which are present at the carboxy terminus of the protein and is referred to as a CaaX motif in which the "C" is cysteine, "a"
is any aliphatic amino acid and "X" is any amino acid. The a subunit is common with a second prenylation enzyme, geranylgeranyl transferase, that has a different (3 subunit and adds a to geranylgeranyl isoprenyl pyrophosphate moiety to the target sequence.
Prenylation is a multistep pathway which includes prenylation of the cysteine residue of the CaaX site, cleavage of the -aaX tripeptide and methylation of the prenyl-cysteine residue. Potentially, each of these steps could represent a target for genetic manipulation of the prenylation process to generate a desired phenotype such as stress tolerance.
In plants, prenylation has been linked to cell cycle control, meristem development, and phytohormone signal transduction, however, few details of the role of prenylation, the substrate proteins or the extent to which the plant system will be analogous to the mammalian and yeast systems are known. The most characterized 2o substrates for CaaX modification are the Ras and a-factor proteins of yeast. Although there are three steps to complete protein maturation, abolition or modification of any one step does not necessarily result in cessation of target biological activities.
Ras function is attenuated if the -aaX tripeptide is not cleaved but not abolished and some proteins retain the -aaX tripeptide after farnesylation.
In Arabidopsis, more than 600 proteins contain a CaaX motif, suggesting a role for the post-translational modification by prenylation in numerous cellular processes. In Arabidopsis, it has been demonstrated that the loss-of function of the (3-subunit of farnesyl transferase will result in a ABA-hypersensitive phenotype. Although it is still not clear why plants lacking the functional (3-subunit of farnesyl transferase become 3o more sensitive to ABA, it clearly suggests that protein prenylation is involved in regulation of the homeostasis of ABA sensitivity. The balance of ABA cellular responses, whether more sensitive or less sensitive to ABA, is possibly regulated by the relative activities of prenylated proteins. The changes in AtCPP expression and gene activity may affect the activity of two pools of genes, one pool acting as positive regulators (pool A) and the second pool (pool B) as negative regulators, which require prenylation in order to function properly. Pool A may contain genes that can promote ABA sensitivity, and pool B genes that may reduce ABA sensitivity. The homeostasis of ABA sensitivity may therefore governed by the ratio of activity of pool A to pool B. For example, in the case of up-regulation of AtCPP in Arabidopsis, the activity ratio of pool A over pool B may be increased due to difference in substrate affinity of pool A proteins toward AtCPP, thus the homeostasis of ABA sensitivity is changed, and the AtCPP over-l0 expression plants are more sensitive to ABA.
This invention is directed at the manipulation of the CaaX prenyl protease enzyme (CPP), which catalyses the proteolytic cleavage of the -aaX tripeptide in the second step of the prenylation process. Included in this invention are vector constructs containing CPP sequence under the control of appropriate regulatory sequences to produce a water-stress tolerant phenotype.
SUMMARY OF THE INVENTION
The present invention is based in part upon the discovery of novel CaaX prenyl protease (CPP) nucleic acid sequences and polypeptides isolated from Arabidopsis thaliana, Brassica napus and Glycine max. The nucleic acids, polynucleotides, proteins and polypeptides, or fragments thereof described herein are collectively referred to as CPP nucleic acids and polypeptides.
Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO:1, SEQ ID N0:14, or SEQ ID
N0:17 or fragment, homolog, analog or derivative thereof. The nucleic acid can include, e.g., a nucleic acid sequence encoding a polypeptide at least 99% identical to a polypeptide that includes the amino acid sequences of SEQ ID N0:2, SEQ ID NO:1 S, or SEQ ID
N0:18 or a nucleic acid sequence encoding a polypeptide at least 96% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:15. In yet another aspect, the invention provides a nucleic acid that includes the sequence of SEQ ID NO: 68, 70, 72 or 74. The nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA
molecule.
Preferably, the nucleic acid is naturally occurring. The invention also provides a nucleic acid sequence that is complementary to the nucleic acid sequence of SEQ ID
NO:1, SEQ
ID N0:14, or SEQ ID N0:17. For example, SEQ ID NO: 16, 19 or 20.
Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein. In various aspects the vector comrprises the nucleic acid sequences of SEQ ID
NO: 4, 5, 36-53.
The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
The invention is also directed to plants and cells transformed with a CPP
nucleic to acid or a vector comprising a CPP nucleic acid. Also included in the invention is the seed, and progeny of the transformed plants or cells.
In a further aspect, the invention includes a substantially purified CPP
polypeptide, e.g., any of the CPP polypeptides encoded by an CPP nucleic acid, and fragments, homologs, analogs, and derivatives thereof. Accordingly, in one aspect, the invention provides an isolated polypeptide molecule that includes the sequence of SEQ
ID N0:2, SEQ ID NO:15, or SEQ ID N0:18.
In yet another aspect the invention provides a polypeptides that includes the sequence of SEQ ID NO: 69, 71, 73 or 75.
In still a further aspect, the invention provides an antibody that binds specifically 2o to an CPP polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof. The invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
The invention also includes a method of producing a transgenic plant which has an altered phenotype such as, but not limited to, increased tolerance to stress, delayed senescence, increased ABA sensitivity, increased yield, increased productivity and increased biomass compared to a wild type plant by introducing into one or more cells of a plant a compound that alters (e.g., increases or decreases) CPP expression or activity in the plant. In one aspect the compound is a CPP nucleic acid or polypeptide. In one 3o emodiment the nucleic acid is an inhibitor or farnesylation. For example, the compound comprises SEQ ID NO: l, 14, 17, 68, 70, 72, 74, 21, 23, 25, 27, 29, 31, 33, 2, 15, 18, 22, 24 26, 28, 30, 32 34, 69, 71, 73, or 75. Alternatively, the compound is a CPP
double stranded RNA-inhibition hair-pin nucleic acid or CPP antisense nucleic acid, such as for example, SEQ ID NO: 16, 19, 20, 5, 35, 37, 42, 45, 46, 48, 49, 51 or 51.

The invention further provides a method for producing a CPP polypeptide by providing a cell containing an CPP nucleic acid, e.g., a vector that includes a CPP
nucleic acid, and culturing the cell under conditions sufficient to express the CPP
polypeptide encoded by the nucleic acid. The expressed CPP polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous CPP
polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The invention is also directed to methods of identifying a CPP polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
to The invention further provides methods of identifying a compound that modulates the activity of a CPP polypeptide by contacting a CPP polypeptide with a compound and determining whether the CPP polypeptide activity is modified.
The invention is also directed to compounds that modulate CPP polypeptide activity identified by contacting a CPP polypeptide with the compound and determining whether the compound modifies activity of the CPP polypeptide, binds to the CPP
polypeptide, or binds to a nucleic acid molecule encoding a CPP polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. is a schematic representation of the vector constructs; A) pBI121-AtCPP, B) pBI121-antisense-AtCPP, C) pBIl21-HP-AtCPP.

Figure 2. is an illustration of (A) nucleic acid and (B) amino acid sequence identities as determined by ClustalW analysis.
Figure 3. is a scan of a typical Southern blot of transgenic Arabidopsis Tl lines carrying the pBI121-AtCPP construct.
Figure 4. is a scan of a typical Southern blot of transgenic Arabidopsis T3 lines carrying the pBI121-HP-AtCPP construct.
Figure 5. is a scan of a typical Southern blot of transgenic Arabidopsis lines carrying the pRD29A-AtCPP construct.
Figure 6. is a scan of a typical Southern blot of transgenic Arabidopsis lines carrying the 1 o pRD29A-HP-AtCPP construct.
Figure 7 is an illustration showing the relative expression of AtCPP mRNA
transcript (solid bars) and AtCPP protein levels (stippled bars) in several pBI121-AtCPP
transgenic lines.
Figure 8. is a histogram showing the percentage of lines which were categorized as 15 ABA sensitive, moderately ABA sensitive or ABA insensitive. Seedlings were assessed on agar plates containing 1 ~M ABA and scored at 21 days growth.
Thirty-six lines of the pBIl21-AtCPP over-expression construct were assessed at 21 days by leaf and seedling development. Thirty-two lines of the 35S-HP-AtCPP down-regulation construct were assessed at 21 days for leaf and seedling 20 development. Each line was assessed by plating approximately 100 seeds per plate and the seedlings scored and recorded as the percent insensitive seedlings per plate. Each line was then expressed as a percent of wild type (Wt). Lines were categorized as sensitive (less than 1% of Wt) solid bars, intermediate (1-50%
of Wt) diagonally lined or insensitive (greater than 50% of Wt) stippled, based on 25 their relationship to Wt and the percentage of each category plotted as a histogram.
Figure 9. is an illustration showing the response of wild type and a pRD29A-HP-AtCPP
transgenic line to various concentrations of ABA in two week old seedlings.
Figure 10. is a histogram showing the analysis of transgenic plants containing the 30 pBI121-AtCPP over-expression construct, (SEQ ID N0:4). Water loss per gram shoot dry weight after four days of water stress treatment. Lines that are marked with a star are those which were strongly ABA sensitive. Lines marked with a triangle are moderately ABA sensitive. Bars represent means of eight replicates.
Lines marked with a filled dot above the bar represents lines which were significantly different from control at a p=0.05 value.
Figure 11. is a histogram showing seed yield in grams of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal water conditions Figure 12. is a bar chart howing growth and yield of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal watering conditions plus a biotic stress condition. Tields as a % of wild type, rosette leaf number, rosette leaf fresh 1o weight and shoot dry weight are plotted.
Figure 13. are photographs showing rowth of transgenic Arabidopsis lines of pBI121-AtCPP grown on agar plates. Changes to root growth visible.
Figure 14. is a bar chart showing rowth of transgenic Arabidopsis lines of pRD29A-HP-AtCPP grown under optimal watering conditions. Rosette leaf number, rosette leaf dry weight and shoot dry weight are plotted.
DETAILED DESCRIPTION OF INVENTION
The present invention provides novel CaaX prenyl protease (CPP) nucleic acid sequences (SEQ ID No:l, SEQ ID N0:14 and SEQ ID N0:17) the encoded polypeptides: SEQ ID N0:2, SEQ ID NO:15 and SEQ ID N0:18) isolated from Arabidopsis thaliana (At) Brassica napus (Bn) and Glycine Max (Gm) respectively. The invention also provides CaaX prenyl protease antisense nucleic acids. (SEQ ID
NO: 16, SEQ ID N0:19 and SEQ ID N0:20). The sequences are collectively referred to as "CPP
nucleic acids", CPP polynucleotides" or "CPP antisense nucleic acids" and the corresponding encoded polypeptide is referred to as a "CPP polypeptide" or "CPP
protein". Unless indicated otherwise, "CPP" is meant to refer to any of the novel sequences disclosed herein. Table A below summarizes the nucleic acids and polypeptides according to the invention Table A
SEQ ID NO. SEQ Type Species Transformed 1 AtCPP NA PCR

2 AtCPP AA Translation 3 At-AFC 1 AA Ref.

4 BI121-AtCPP NA ConstructAt, Bn BI121-HP-AtCPP NA ConstructAt 6 AtCPP BamFW NA Primer 7 AtCPP SmaRV NA Primer 8 AtCPP-HP-SacFW NA Primer 9 AtCPP-HP-SacRV NA Primer BI121-AtCPP ForwardNA Primer I1 BI121-antiAtCPP-SmaFWNA Primer 12 BI121-antiAtCPP-BamRVNA Primer 13 35S-HP-AtCPP ReverseNA Primer 14 BnCPP NA PCR

BnCPP AA Translation 16 BnCPP antisense NA PCR

17 GmCPP NA PCR

18 GmCPP AA Translation 19 GmCPP antisense NA PCR

AtCPP antisense NA PCR

21 BASF-AT1 NA Ref.

22 BASF-AT1 AA Ref.

23 BASF-AT2 NA Ref.

24 BASF-AT2 AA Ref.

BASF-Corn NA Ref.

26 BASF-Corn AA Ref.

27 BASF-So NA Ref.

28 BASF-So AA Ref.

29 AFC 1 NA Ref.

AFC1 AA Ref.

31 AT4g01320 NA Ref.

32 AT4 01320 AA Ref.

33 AF007269 NA Ref.

34 AF007269 AA Ref.

BI121-antisense-AtCPPNA Construct 36 RD29A-AtCPP NA ConstructAt, Bn 37 RD29A-HP-AtCPP NA ConstructAt 38 RD29A-antisense-AtCPPNA Construct 39 MuA-AtCPP NA ConstructGm, Zm MuA-GmCPP NA Construct 41 BI121-GmCPP Construct 42 BI121-HP-GmCPP Construct 43 BI121-antisense-GmCPP Construct 44 RD29A-GmCPP Construct RD29A-HP-GmCPP Construct 46 RD29A-antisense-GmCPP Construct 47 BI121-BnCPP Construct 48 BI121-HP-BnCPP Construct 49 BI121-antisense-BnCPP Construct RD29A-BnCPP Construct 51 RD29A-HP-BnCPP Construct 52 RD29A-antisense-BnCPP Construct 53 MuA-BnCPP Construct 54 GmCPP SmaFW Primer GmCPP SacRV Primer 56 BnCPP-anti-SmaFW Primer 57 BnCPP-anti-BamRV Primer 58 BnCPP-HP-Sac-FW Primer 59 BnCPP-HP-Sac-RV Primer 60 BnCPP-HP-BamFW Primer 61 BnCPP-HP-XbaRV Primer 62 GmCPP-HP-Sac-FW Primer 63 GmCPP-HP-Sac-RV Primer 64 GmCPP-HP-BamFW Primer 65 GmCPP-HP-XbaRV Primer 66 RD29AP Primer 67 Nosterm-RV Primer 68 Consensus- BASF NA

69 Consensus- BASF AA

70 Consensus- GenericNA

71 Consensus- GenericAA

72 Consensus- PPI NA

73 Consensus- PPI AA

74 Consensus- PPI/GenericNA

75 Consensus- PPI/GenrericAA

In a BLAST search of public sequence databases, it was found, for example, that the Arabidopsis thaliana nucleic acid sequence has 99.5 % identity to an Arabidopsis thaliana CaaX processing zinc-metallo endoprotease (AFC1) mRNA
(Genbank Accesion No.: AF353722). The full amino acid sequence of the protein of the invention was found to be 98.8 % identical to Arabidopsis thaliana CaaX
processing zinc-metallo endoprotease (AFC1) polypeptide (Genbank Accesion No.:AAK39514).
A
ClustalW alignment of the Arabidopsis thaliana CPP polypeptide (SEQ ID N0:2), the Brassica napus CPP polypeptide (SEQ ID NO:15), the Glycine max CPP polypeptide to (SEQ ID N0:18) and seven other published CPP sequences is illustrated in Table 6B.
ClustalW alignment of these polypeptides indicate that SEQ ID N0:2, SEQ ID
NO:15 and SEQ ID N0:18 are 99%, 93% and 83% identical to the published AFC sequence (SEQ ID N0:30) respectively. The Glycine max CPP polypeptide (SEQ ID N0:18) is 99% identical to the published sequence shown as SEQ ID N0:28. Similarly, ClustalW
15 alignment of the Arabidopsis thaliana CPP polynucleotide (SEQ ID NO:1 ), the Brassica napus CPP polynucleotide (SEQ ID N0:14), the Glycine max CPP polynucleotide (SEQ
ID N0:17) and seven other published CPP sequences is illustrated in Table 6a indicate that SEQ ID NO:1, SEQ ID N0:14 and SEQ ID N0:17 are 99%, 93% and 77% identical to the published AFC sequence (SEQ ID N0:30) respectively. The Glycine max CPP
2o polynucleotide (SEQ ID N0:17) is 93% identical to the published sequence shown as SEQ ID N0:27.
CaaX prenyl proteases belong to a family of putative membrane-bound proteins that are involved in protein and/or peptide modification (i. e., prenylation) and secretion.
Prenylation is a post translational modification of specific proteins and is required for the proper localization of these polypeptides to the correct cellular site for functionality.
Prenylation is a three step process involving the addition of either a C 15 farnesyl, or C20 geranylgeranyl group to the cysteine residue of the target 3' terminal CaaX
sequence, where "C" is a cysteine, "a" is any aliphatic amino acid and "X" is any amino acid.
Secondly, a CaaX prenyl protease (CPP) cleaves the -aaX tripeptide from the protein and thirdly the exposed a-carboxyl group of the cysteine is methylated by a prenyl-cysteine methyltransferase.
Protein farnesylation, the addition of a C-terminal, 1 S carbon chain to protein and subsequent processing is a three step enzymatic reaction including farnesylation, l0 proteolytic cleavage and methylation. First, a farnesyltransferase adds the C-terminal 15 carbon chain to a conserved cysteine residue of the CaaX terminal motif, where "C" is a Cystine, "a" is an aliphatic amino acid and "X" is any amino acid. Second, the last three amino acid residues (aaX) are cleaved by a prenyl protease. Lastly, the modified cysteine is methylated by a methylase to create the final active product of the protein farnesylation pathway. The Applicant's have shown previously that over expression and down-regulation of the alpha or the beta farnesyl transferase gene in plant cells ( i.e, the first step in farnesylation) results in plants with an altered phenotype such as but not limited to drought tolerance and delayed senescence. The present invention shows that over expression and down-regulation of the prenyl protease gene (i.e, the second step in farnesylation) in plant cells also results in a plant displaying an altered phenotype including for example but not limited to drought tolerance and increased resistance to biotic and abiotic stress. These results taken together support the hypothesis that modification of the expression of any of the enzymes in the farnnesylation pathway in a plant cell will result in a plant displaying an altered phenotype Based on their structural and functional relatedness to known CaaX prenyl protease proteins, the CPP proteins are novel members of the CaaX prenyl protease family of proteins. CPP nucleic acids, and their encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, the nucleic 3o acids (i.e., sense or antisense CPP nucleic acids) can be used produce transgenic plants that have an increase resistance to biotic and abiotic stresses, e.g., chilling stress, salt stress, water stress, wound healing, pathogen challenge, or herbicides.
Additionally, the transgenic plants have an increased productivity during both optimal and suboptimal growth conditions, increased yield, or increased biomass. Alternatively, the transgenic plants have an increased sensitivity to the phytohormone abscisic acid (ABA).
This invention includes methods to up-regulate the CPP enzyme activity in transgenic plants, cells and tissue cultures by using an over-expression vector construct and methods to down-regulate the CPP enzyme activity in transgenic plants, cells and tissue cultures by using a double stranded RNA-inhibition, hairpin vector constructs or antisense constructs. Alteration (i.e., upregulation or downregulation) of CPP
enzyme activity or expression results in transgenic plants with altered phenotypes as described below. These methods are by way of example to produce the up-regulation or down-1 o regulation effects and are not meant to be limiting as to the method of achieving this outcome.
Additionally, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, CPP
activity. Alternatively, the CPP nucleic acids and polypeptides can be used to identify proteins that are members of the CaaX prenyl protease family of proteins.
Additional utilities for CPP nucleic acids and polypeptides according to the invention are disclosed herein.
CPP Nucleic Acids The nucleic acids of the invention include those that encode a CPP polypeptide or 2o protein. As used herein, the terms polypeptide and protein are interchangeable.
In some embodiments, a CPP nucleic acid encodes a mature CPP polypeptide. As used herein, a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein.
The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene.
Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises.
Examples of 3o such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence.

Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N
remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature"
form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature to polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
Among the CPP nucleic acids is the nucleic acid whose sequence is provided in SEQ ID NO: l, SEQ ID N0:14 OR SEQ ID N0:17 or a fragment thereof.
Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO: 1, SEQ ID
N0:14 OR SEQ ID N0:17 or a fragment thereof, any of whose bases may be changed from the corresponding base shown in SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17, while still encoding a protein that maintains at least one of its CPP-like activities and physiological functions. The invention further includes the complement of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17, including 2o fragments, derivatives, analogs and homologs thereof. Complement nucleic acid CPP
sequences include SEQ ID NO: 16, 19 or 20. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
One aspect of the invention pertains to isolated nucleic acid molecules that encode CPP proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify CPP-encoding nucleic acids (e.g., CPP mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of CPP nucleic acid molecules.
As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., 3o cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

"Probes" refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers.
Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
Examples of l0 isolated nucleic acid molecules include, but are not limited to, recombinant DNA
molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated CPP nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID N0:14, or SEQ ID
N0:17or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17 as a hybridization probe, CPP nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2°d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., eds., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to CPP nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
l0 Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1, 14 or 17, or a complement thereof. Oligonucleotides may be chemically synthesized and may be used 15 as probes.
In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is 2o a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17, or a portion of these nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID
N0:14 or SEQ ID N0:17 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: l, SEQ ID N0:14 or SEQ ID N0:17 that it can hydrogen bond 25 with little or no mismatches to the nucleotide sequence shown in SEQ ID NO:
1, SEQ ID
N0:14 or SEQ ID N0:17, thereby forming a stable duplex. Exemplary complement nucleic acid sequences include the sequences of SEQ ID NO: 16, 19 or 20.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term "binding"
30 means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17, e.g., s a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of CPP. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less 1 o than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but 15 differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
20 Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an 25 aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS nr MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and 30 below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by reference in its entirety). A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a CPP polypeptide.
Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
Exemplary homologous nucleic acid sequences include the nucleic acid sequences of SEQ ID NO: 68, 70, 72 and 74. Homologous nucleic acid sequences include those l0 nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID N0:2, SEQ ID N0:15 and SEQ ID N0:18 , as well as a polypeptide having CPP
activity, e.g. substrate binding.
The nucleotide sequence determined from the cloning of the Arabidopsis thaliana, Brassica napus or Glycine max CPP gene allows for the generation of probes and primers designed for use in identifying and/or cloning CPP homologues in other cell types, e.g., from other tissues, as well as CPP homologues from other plants.
The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: l, SEQ
ID
N0:14 or SEQ ID N0:17; or an anti-sense strand nucleotide sequence of SEQ ID
NO: 1, SEQ ID N0:14 or SEQ ID N0:17; or of a naturally occurring mutant of SEQ ID NO:
1, SEQ ID N0:14 or SEQ ID N0:17.
Probes based on the Arabidopsis thaliana, Brassica napus or Glycine max CPP
nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CPP
protein, such as by measuring a level of a CPP-encoding nucleic acid in a sample of cells from a subject e.g., detecting CPP mRNA levels or determining whether a genomic CPP gene has been mutated or deleted.
A "polypeptide having a biologically active portion of CPP" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically active portion of CPP" can be prepared by isolating a portion of SEQ ID
NO: 1, SEQ ID N0:14 or SEQ ID N0:17 that encodes a polypeptide having a CPP
biological activity (biological activities of the CPP proteins are described below), expressing the encoded portion of CPP protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of CPP. In another embodiment, a nucleic acid fragment encoding a biologically active portion of CPP includes one or more regions.
CPP Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17 due to the degeneracy of the genetic code. These nucleic acids thus encode the same CPP
protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, SEQ
ID
N0:14 or SEQ ID N0:17, e.g., the polypeptide of SEQ ID NO: 2, SEQ ID N0:15, SEQ
ID NO: 18. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO:15, SEQ ID NO: 18.
2o In addition to the Arabidopsis thaliana, Brassica napus or Glycine max CPP
nucleotide sequence shown in SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID N0:17, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of CPP may exist within a population (e.g., the plant). Such genetic polymorphism in the CPP gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a CPP protein, preferably a plant CPP protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the CPP
gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in CPP that are the result of natural allelic variation and that do not alter the functional activity of CPP are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding CPP proteins from other species, and thus that have a nucleotide sequence that differs from the sequence of SEQ ID
NO: 1, SEQ ID N0:14 or SEQ ID N0:17 are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants and homologues of the CPP cDNAs of the invention can be isolated based on their homology to the Arabidopsis thaliana, Brassica napus or Glycine max CPP nucleic acids disclosed herein using the cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ
ID N0:14 or SEQ ID N0:17. In another embodiment, the nucleic acid is at least 10, 25, l0 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%
homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding CPP proteins derived from species other than Arabidopsis thaliana, Brassica napus or Glycine max) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different depending upon circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for 1s longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, SEQ ID N0:14 or SEQ ID
N0:17 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ
ID
N0:14 or SEQ ID N0:17, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency 2o hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS
and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID N0:14, or SEQ ID NO: 17 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH
7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1 % SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
Conservative mutations In addition to naturally-occurring allelic variants of the CPP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, SEQ ID
N0:14 I o or SEQ ID NO: 17, thereby leading to changes in the amino acid sequence of the encoded CPP protein, without altering the functional ability of the CPP
protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential"
amino acid residues can be made in the sequence of SEQ ID NO: l, SEQ ID N0:14 or SEQ ID NO: 17. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CPP without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the CPP proteins of the present invention, are predicted to be particularly unamenable to alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding CPP
2o proteins that contain changes in amino acid residues that are not essential for activity.
Such CPP proteins differ in amino acid sequence from SEQ ID NO: 2, SEQ ID
NO:15 or SEQ ID N0:18, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:1 S or SEQ ID N0:18. Preferably, the protein encoded by the nucleic acid is at least about 80% homologous to S SEQ ID NO:
2, SEQ
ID NO:15 or SEQ ID N0:18 more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2, SEQ ID NO:15 or SEQ
ID
N0:18.
An isolated nucleic acid molecule encoding a CPP protein homologous to the protein of SEQ ID NO: 2, SEQ ID NO:1 S or SEQ ID N0:18 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 or SEQ ID N0:14, or SEQ ID N0:17 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, SEQ
ID N0:14 or SEQ ID N0:17 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with to basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in CPP is replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CPP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CPP biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: l, SEQ ID N0:14 or SEQ ID N0:17 the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant CPP protein can be assayed for (1) the ability to form protein:protein interactions with other CPP proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant CPP
protein and a CPP receptor; (3) the ability of a mutant CPP protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins);
(4) the ability to bind CPP protein; or (5) the ability to specifically bind an anti-CPP
protein antibody.
Antisense CPP Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID N0:14 or SEQ ID

N0:17 , or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire CPP coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a CPP protein of SEQ ID NO: 2 or SEQ ID NO:15 or SEQ ID N0:18 or antisense nucleic acids complementary to a CPP nucleic acid sequence of SEQ ID
NO: 1, to SEQ ID N0:14 or SEQ ID N0:17 are additionally provided. Exemplary CPP anti-sense nucleic acid include the nucleic acid sequences of SEQ ID N0:16, 19, and 20.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding CPP. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of Arabidopsis thaliana, Brassica napus or Glycine max CPP corresponds to SEQ ID NO: 2 or SEQ
ID
NO:15 or SEQ ID NO:l 8). In another embodiment, the antisense nucleic acid molecule is antisense to a "noricoding region" of the coding strand of a nucleotide sequence encoding CPP. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding CPP disclosed herein (e.g., SEQ ID
NO: 1 or SEQ ID N0:14 or SEQ ID N0:17), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of CPP mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of CPP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CPP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or SO nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of 2o an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CPP
protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641 ). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (moue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (moue et al. (1987) FEBS Lett 215: 327-330).
Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in applications.
Double Stranded RNA Inhibition (RNAi) by Hairpin Nucleic Acids Another aspect of the invention pertains to the use of post transcriptional gene 2o silencing (PTGS) to repress gene expression. Double stranded RNA can initiate the sequence specific repression of gene expression in plants and animals. Double stranded RNA is processed to short duplex oligomers of 21-23 nucleotides in length.
These small interfering RNA's suppress the expression of endogenous and heterologous genes in a sequence specific manner (Fire et al. Nature 391:806-811, Carthew, Curr. Opin.
in Cell Biol., 13:244-248, Elbashir et al., Nature 411:494-498). A RNAi suppressing construct can be designed in a number of ways, for example, transcription of a inverted repeat which can form a long hair pin molecule, inverted repeats separated by a spacer sequence that could be an unrelated sequence such as GUS or an intron sequence.
Transcription of sense and antisense strands by opposing promoters or cotranscription of sense and antisense genes.
CPP Ribozymes and PNA moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CPP mRNA transcripts to thereby inhibit translation of CPP mRNA. A ribozyme having specificity for a CPP-encoding nucleic acid can be designed based upon the nucleotide sequence of a CPP DNA disclosed herein (i. e., SEQ ID NO: 1, SEQ ID N0:14 or SEQ
ID N0:17). For example, a derivative of a Tetrahymena L-19 IVS RNA can be l0 constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CPP-encoding mRNA. See, e.g., Cech et al. U.S.
Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CPP mRNA
can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, CPP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CPP (e.g., the CPP
promoter and/or enhancers) to form triple helical structures that prevent transcription of the CPP
gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6:
569-84;
Helene. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:
807-15.
In various embodiments, the nucleic acids of CPP can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. ( 1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS
93:
14670-675.
PNAs of CPP can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of CPP can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.
(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.
(1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of CPP can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug 1 o delivery known in the art. For example, PNA-DNA chimeras of CPP can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) 2o amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al.
(1975) Bioorg Med Chem Lett 5: 1119-11124.
CPP Polypeptides A CPP polypeptide of the invention includes the protein whose sequence is provided in SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID N0:18. The invention also includes a mutant or variant protein any of whose residues may be changed from the 3o corresponding residue shown in SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID N0:18 while still encoding a protein that maintains its CPP-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more of the residues may be so changed in the mutant or variant protein. In some embodiments, the CPP polypeptide according to the invention is a mature polypeptide.

In general, a CPP -like variant that preserves CPP-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated CPP proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also to provided are polypeptide fragments suitable for use as immunogens to raise anti-CPP
antibodies. In one embodiment, native CPP proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, CPP proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a CPP protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the CPP protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of CPP protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of CPP protein having less than about 30% (by dry weight) of non-CPP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-CPP protein, still more preferably less than about 10% of non-CPP protein, and most preferably less than about 5% non-CPP
protein.
When the CPP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of CPP protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"

includes preparations of CPP protein having less than about 30% (by dry weight) of chemical precursors or non-CPP chemicals, more preferably less than about 20%
chemical precursors or non-CPP chemicals, still more preferably less than about 10%
chemical precursors or non-CPP chemicals, and most preferably less than about 5%
chemical precursors or non-CPP chemicals.
Biologically active portions of a CPP protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the CPP protein, e.g., the amino acid sequence shown in SEQ ID NO: 2 that include fewer amino acids than the full length CPP proteins, and exhibit at least one activity of a to CPP protein, e.g. substrate binding. Typically, biologically active portions comprise a domain or motif with at least one activity of the CPP protein. A biologically active portion of a CPP protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a CPP protein of the present invention may contain at least one of the above-identified domains conserved between the CPP
proteins, e.g.. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native CPP protein.
A biologically active portion or a CPP protein can be the N-terminal domain of the CPP polypeptide. Alternatively, a biologically active portion or a CPP
protein can be the C-terminal domain of the CPP polypeptide. Preferably, the biologically active portion comprises at least 75 amino acids of the C- terminal domain. More preferably, the biologically active portion comprises at least 25 amino acids of the C-terminal domain. Most preferably, the biologically active portion comprises at least 10 amino acids of the C- terminal.
In an embodiment, the CPP protein has an amino acid sequence shown in SEQ ID
NO: 2, SEQ ID NO:15 or SEQ ID N0:18. In other embodiments, the CPP protein is substantially homologous to SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID N0:18 and retains the functional activity of the protein of SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID
3o N0:18, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the CPP
protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of S SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID N0:18 and retains the functional activity of the CPP proteins of SEQ ID NO: 2, SEQ ID NO:15 or SEQ ID
N0:18.
Exemplary homologous CPP polypeptides include for example the polypeptide sequences of SEQ ID NO: 69, 71, 73 and 75.
Determining homology between two or more sequence To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either of the sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package.
See, Needleman and Wunsch 1970 JMoI Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 2o and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA
sequence shown in SEQ ID NO:1 or SEQ ID N0:14 or SEQ ID N0:17.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. The term "percentage of positive residues" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i. e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
Chimeric and fusion proteins The invention also provides CPP chimeric or fusion proteins. As used herein, a CPP "chimeric protein" or "fusion protein" comprises a CPP polypeptide operatively linked to a non-CPP polypeptide. An "CPP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to CPP, whereas a "non-CPP polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the CPP protein, e.g., a protein that is different from the CPP protein and that is derived from the same or a different organism. Within a CPP
fusion protein the CPP polypeptide can correspond to all or a portion of a CPP
protein.
In one embodiment, a CPP fusion protein comprises at least one biologically active portion of a CPP protein. In another embodiment, a CPP fusion protein comprises at least two biologically active portions of a CPP protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the CPP polypeptide and the non-CPP polypeptide are fused in-frame to each other. The non-CPP polypeptide can be fused to the N-terminus or C-terminus of the CPP polypeptide.
A CPP chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CPP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CPP protein.
CPP agonists and antagonists The present invention also pertains to variants of the CPP proteins that function as either CPP agonists (mimetics) or as CPP antagonists. Variants of the CPP
protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the CPP
protein. An agonist of the CPP protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the CPP protein.
An antagonist of the CPP protein can inhibit one or more of the activities of the naturally occurring form of the CPP protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the CPP
protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function.
Variants of the CPP protein that function as either CPP agonists (mimetics) or as 2o CPP antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the CPP protein for CPP protein agonist or antagonist activity. In one embodiment, a variegated library of CPP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A
variegated library of CPP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CPP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of CPP sequences therein. There are a variety of methods which can be used to produce libraries of potential CPP variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CPP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
Polypeptide libraries In addition, libraries of fragments of the CPP protein coding sequence can be used to generate a variegated population of CPP fragments for screening and subsequent selection of variants of a CPP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CPP
coding sequence with a nuclease under conditions wherein nicking occurs only about once per l0 molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the CPP protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CPP
proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CPP variants (Arkin and Yourvan (1992) PNAS
89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
CPP Antibodies CPP polypeptides, including chimeric polypeptides, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens to generate antibodies that immunospecifically-bind these peptide components. Such antibodies include, e.g., polyclonal, monoclonal, chimeric, single chain, Fab fragments and a Fab expression library. In a specific embodiment, fragments of the CPP polypeptides are used as immunogens for antibody production. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to a CPP
polypeptides, or derivative, fragment, analog or homolog thereof.
For the production of polyclonal antibodies, various host animals may be immunized by injection with the native peptide, or a synthetic variant thereof, or a derivative of the foregoing. Various adjuvants may be used to increase the immunological response and include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, to polyanions, peptides, oil emulsions, dinitrophenol, etc.) and human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum.
For preparation of monoclonal antibodies directed towards a CPP polypeptides, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
Such techniques include, but are not limited to, the hybridoma technique (see, Kohler and Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see, Kozbor, et al., 1983. Immunol Today 4: 72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see, Cole, et al., 1985.
In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Human 2o monoclonal antibodies may be utilized in the practice of the present invention and may be produced by the use of human hybridomas (see, Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see, Cole, et al., 1985. In: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., pp. 77-96).
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a CPP polypeptides (see, e.g., U.S. Patent No.
4,946,778). In addition, methodologies can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for 3o a CPP polypeptides or derivatives, fragments, analogs or homologs thereof.
Antibody fragments that contain the idiotypes to a CPP polypeptides may be produced by techniques known in the art including, e.g., (i) an F(ab')z fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
In one embodiment, methodologies for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, 'selection of antibodies that are specific to a particular domain of a CPP polypeptides is facilitated by generation of hybridomas that bind to the fragment of a CPP polypeptides possessing such a domain. Antibodies that are specific for a domain within a CPP polypeptides, or derivative, fragments, analogs or homologs thereof, are 1o also provided herein. The anti-CPP polypeptide antibodies may be used in methods known within the art relating to the localization and/or quantitation of a CPP
polypeptide(e.g., for use in measuring levels of the peptide within appropriate physiological samples, for use in diagnostic methods, for use in imaging the peptide, and the like).
CPP Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a CPP protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule 2o capable of transporting another nucleic acid to which it has been linked.
Exemplary expression vector constructs include for example the constructs of SEQ ID NO:
4" 5, 36, 37, 39, 40, 441, 42, 44, 45, 47, 48, 50 , 51 and 53. Additional exemplary expression vector constructs include contructs comprising CPP anti-sense nucleic acid such as SEQ
ID NO: 38. 43., 46, 49, 52. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication).
Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked.
Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of to interest is linked to the regulatory sequences) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory IS sequences are described, for example, in Goeddel, GENE EXPRESSION
TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Examples of suitable 20 promoters include for example constitutive promoters, ABA inducible promoters, tissue specific promters or guard cell specific promoters. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
The expression vectors of the invention can be introduced into host cells to thereby 25 produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CPP proteins, mutant forms of CPP proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of CPP proteins in prokaryotic or eukaryotic cells. For example, CPP
30 proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells, plant cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
1o Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 1 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20:
3o 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the CPP expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, CPP can be expressed in insect cells using baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 s cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
1o EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e. g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY
I5 MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In yet another embodiment, a nucleic acid of the invention is expressed in plants cells using a plant expression vector. Examples of plant expression vectors systems include tumor inducing (Ti) plasmid or portion thereof found in Agrobacterium, 2o cauliflower mosaic virus (CAMV) DNA and vectors such as pBI121 .
For expression in plants, the recombinant expression cassette will contain in addition to the CPP nucleic acids, a plant promoter region, a transcription initiation site (if the coding sequence to transcribed lacks one), and a transcription termination/polyadenylation sequence. The termination/polyadenylation region may be 25 obtained from the same gene as the promoter sequence or may be obtained from different genes. Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
Examples of suitable promotors include promoters from plant viruses such as the 35S
promoter from cauliflower mosaic virus (CaMV). Odell, et al., Nature, 313: 810-30 (1985). and promoters from genes such as rice actin (McElroy, et al., Plant Cell, 163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol. Biol., 12: 619-632 (1992);
and Christensen, et al., Plant Mol. Biol., 18: 675-689 (1992)); pEMU (Last, et al., Theor.
Appl. Genet., 81: 581-588 (1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730 (1984));
maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231: 276-285 (1992); and Atanassvoa, et al., Plant Journal, 2(3): 291-300 (1992)), the 5'- or 3'-promoter derived from T-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), a synthetic promoter, s such as, Rsyn7, SCP and UCP promoters, ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heat shock promoters, seed-specific promoters and other transcription initiation regions from various plant genes, for example, include the various opine initiation regions, such as for example, octopine, mannopine, and nopaline.
Additional regulatory elements that may be connected to a CPP encoding nucleic acid l0 sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell. Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements CPP gene are known, and include, but are not limited to, 3' termination and/or polyadenylation 15 regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinase inhibitor II
(PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650 (1986) and hereby incorporated by reference); and An" et al., Plant Cell, 1: 115-122 (1989));
and the CaMV
19S gene (Mogen, et al., Plant Cell, 2: 1261-1272 (1990)).
20 Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264: 4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene (DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides which target proteins to the vacuole like the sweet potato sporamin gene 25 (Matsuka, et al., Proc. Nat'1 Acad. Sci. (USA), 88: 834 (1991)) and the barley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), or signals which cause proteins to be secreted such as that of PRIb (Lind, et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which target proteins to the plastids such as that of rapeseed enoyl-ACP
reductase (Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful in the invention.
3o In another embodiment, the recombinant expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Especially useful in connection with the nucleic acids of the present invention are expression systems which are operable in plants.
These include systems which are under control of a tissue-specific promoter, as well as those which involve promoters that are operable in all plant tissues.
Organ-specific promoters are also well known. For example, the patatin class I
promoter is transcriptionally activated only in the potato tuber and can be used to target gene expression in the tuber (Bevan, M., 1986, Nucleic Acids Research 14:4625-4636).
Another potato-specific promoter is the granule-bound starch synthase (GBSS) promoter (Visser, R.G.R, et al., 1991, Plant Molecular Biology 17:691-699).
Other organ-specific promoters appropriate for a desired target organ can be isolated 1 o using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, P., 1986, Trans. R. Soc. London B314:343).
For in situ production of the antisense mRNA of GST, those regions of the GST
gene which are transcribed into GST mRNA, including the untranslated regions thereof, are inserted into the expression vector under control of the promoter system in a reverse orientation. The resulting transcribed mRNA is then complementary to that normally produced by the plant.
The resulting expression system or cassette is ligated into or otherwise 2o constructed to be included in a recombinant vector which is appropriate for plant transformation. The vector may also contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to 6418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct.
Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobacterium transformations, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
to Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation"
and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e., express) a polypeptide of the invention encoded in a an open reading frame of a polynucleotide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been 2o introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
A number of types of cells may act as suitable host cells for expression of a polypeptide encoded by an open reading frame in a polynucleotide of the invention.
Plant host cells include, for example, plant cells that could function as suitable hosts for the expression of a polynucleotide of the invention include epidermal cells, mesophyll and other ground tissues, and vascular tissues in leaves, stems, floral organs, and roots from a variety of plant species, such as Arabidopsis thaliana, Nicotiana tabacum, Brassica napus, Zea mays, Oryza sativa, Gossypium hirsutum and Glycine max.
Alternatively, it may be possible to produce a polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional polypeptide, if the polypeptide is of sufficient length and conformation to have activity. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
A polypeptide may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed polypeptide or protein may then be purified from such culture (e.g., from culture medium to or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the polypeptide or protein may also include an affinity column containing agents which will bind to the protein;
one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue 3GA Sepharose~; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, a polypeptide or protein may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein containing a six-residue histidine tag. The histidine-tagged protein will then bind to a Ni-affinity column. After elution of all other proteins, the histidine-tagged protein can be eluted to achieve rapid and efficient purification. One or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide. The protein or polypeptide thus purified is substantially free of other plant proteins or polypeptides and is defined in accordance with the present invention as "isolated."
Transformed Plants Cells and Transgenic Plants The invention includes protoplast, plants cells, plant tissue and plants (e.g., monocots and dicots transformed with a CPP nucleic acid (i. e, sense or antisense), a vector containing a CPP nucleic acid (i. e, sense or antisense)or an expression vector containing a CPP nucleic acid (i. e, sense or antisense). As used herein, "plant" is meant to include not only a whole plant but also a portion thereof (i. e., cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds).
The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Gossypium, Picea, Caco, and Populus.
In some aspects of the invention, the transformed plant is resistant to biotic and abiotic stresses, e.g., chilling stress, salt stress, water stress (e.g., drought), disease, grazing pests and wound healing. Additionally, the invention also includes a transgenic plant that is resistant to pathogens such as for example fungi, bacteria, nematodes, viruses and parasitic weeds. Alternatively, the transgenic plant is resistant to herbicides or has delayed senesence. The transgenic plant has an increase in yield, productivity, biomass or ABA sensitivity. By resistant is meant the plant grows under stress conditions (e.g., high salt, decreased water, low temperatures) or under conditions that normally inhibit, to some degree, the growth of an untransformed plant.
Methodologies to determine plant growth or response to stress include for example, height measurements, weight meaurements, leaf area, ability to flower, water use, transpiration rates and yield.
The invention also includes cells, tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds and the progeny derived from the tranformed plant.
Numerous methods for introducing foreign genes into plants are known and can be used to insert a gene into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., (1993) "Procedure for Introducing Foreign DNA into Plants", In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-and Andrew Bent in, Clough SJ and Bent AF, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, polyethylene glycol (PEG) transformation, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)), electroporation, protoplast transformation, micro-injection, flower dipping and biolistic bombardment.
Agrobacterium-mediated Transformation The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A.
1 o rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genes responsible for genetic transformation of plants. See, for example, Kado, Crit. Rev.
Plant Sci., 10: 1-32 (1991). Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber et al., supra;
15 and Moloney, et al, Plant Cell Reports, 8: 238-242 (1989).
Transgenic Arabidopsis plants can be produced easily by the method of dipping flowering plants into an Agrobacterium culture, based on the method of Andrew Bent in, Clough SJ and Bent AF, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Wild type plants are grown until the 2o plant has both developing flowers and open flowers. The plant are inverted for 1 minute into a solution of Agrobacterium culture carrying the appropriate gene construct. Plants are then left horizontal in a tray and kept covered for two days to maintain humidity and then righted and bagged to continue growth and seed development. Mature seed is bulk harvested.
25 Direct Gene Transfer A generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 mu.m. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which 30 is sufficient to penetrate the plant cell walls and membranes. (Sanford, et al., Part. Sci.
Technol., 5: 27-37 (1987); Sanford, Trends Biotech, 6: 299-302 (1988);
Sanford, Physiol. Plant, 79: 206-209 (1990); Klein, et al., Biotechnology, 10: 286-291 (1992)).

Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang, et al., BioTechnology, 9: 996-996 (1991). Alternatively, liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, for example, Deshayes, et al., EMBO J., 4: 2731-2737 (1985); and Christou, et al., Proc.
Nat'1. Acad. Sci. (USA), 84: 3962-3966 (1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. See, for example, Hain, et al., Mol. Gen. Genet., 199: 161 (1985);
and Draper, et al., Plant Cell Physiol., 23: 451-458 (1982).
Electroporation of protoplasts and whole cells and tissues has also been to described. See, for example, Donn, et al., (1990) In: Abstracts of the VIIth Int;l.
Congress on Plant Cell and Tissue Culture IAPTC, A2-38, page 53; D'Halluin et al., Plant Cell, 4: 1495-1505 (1992); and Spencer et al., Plant Mol. Biol., 24: 51-61 (1994).
Particle Wounding/Agrobacterium Delivery Another useful basic transformation protocol involves a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA
delivery, as described by Bidney, et al., Plant Mol. Biol., 18: 301-31 (1992).
Useful plasmids for plant transformation include Bin 19. See Bevan, Nucleic Acids Research, 12: 8711-8721 (1984), and hereby incorporated by reference.
2o In general, the intact meristem transformation method involves imbibing seed for 24 hours in the dark, removing the cotyledons and root radical, followed by culturing of the meristem explants. Twenty-four hours later, the primary leaves are removed to expose the apical meristem. The explants are placed apical dome side up and bombarded, e.g., twice with particles, followed by co-cultivation with Agrobacterium. To start the co-cultivation for intact meristems, Agrobacterium is placed on the meristem.
After about a 3-day co-cultivation period the meristems are transferred to culture medium with cefotaxime plus kanamycin for the NPTII selection.
The split meristem method involves imbibing seed, breaking of the cotyledons to produce a clean fracture at the plane of the embryonic axis, excising the root tip and then 3o bisecting the explants longitudinally between the primordial leaves. The two halves are placed cut surface up on the medium then bombarded twice with particles, followed by co-cultivation with Agrobacterium. For split meristems, after bombardment, the meristems are placed in an Agrobacterium suspension for 30 minutes. They are then removed from the suspension onto solid culture medium for three day co-cultivation.

After this period, the meristems are transferred to fresh medium with cefotaxime plus kanamycin for selection.
Transfer by Plant Breeding Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conventional plant breeding methods can be used to transfer the gene and associated regulatory sequences via crossing and backcrossing.
Such intermediate methods will comprise the fiu-ther steps of: ( 1 ) sexually crossing the transgenic plant with a plant from a second taxon; (2) recovering reproductive material l0 from the progeny of the cross; and (3) growing transgenic plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the second taxon can be substantially preserved by expanding this method to include the further steps of repetitively: (1) backcrossing the transgenic progeny with non-transgenic plants from the second taxon; and (2) selecting for expression of an associated marker gene among the progeny of the backcross, until the desired percentage of the characteristics of the second taxon are present in the progeny along with the gene or genes imparting marker gene trait.
By the term "taxon" herein is meant a unit of botanical classification. It thus includes, genus, species, cultivars, varieties, variants and other minor taxonomic groups which 2o lack a consistent nomenclature.
Regeneration of Transformants The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988).
This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983). In particular, U.S. Pat. No.
5,349,124 (specification incorporated herein by reference) details the creation of genetically transformed lettuce cells and plants resulting therefrom which express hybrid crystal proteins conferring insecticidal activity against Lepidopteran larvae to such plants.
This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.
1o Preferably, the regenerated plants are self pollinated to provide homozygous transgenic plants, or pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants.
A transgenic plant of the present invention containing a desired polypeptide is cultivated using 15 methods well known to one skilled in the art.
A preferred transgenic plant is an independent segregant and can transmit the CPP gene and its activity to its progeny. A more preferred transgenic plant is homozygous for the gene, and transmits that gene to all of its offspring on sexual mating.
Seed from a transgenic plant may be grown in the field or greenhouse, and resulting 2o sexually mature transgenic plants are self pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for increased expression of the CPP transgene.
Method of Producing Transgenic Plants Also included in the invention are methods of producing a transgenic plant.
The 25 method includes introducing into one or more plant cells a compound that alters CaaX
prenyl protease expression or activity in the plant to generate a transgenic plant cell and regenerating a transgenic plant from the transgenic cell. In some aspects the compound increases alters CaaX prenyl protease expression or activity. Alternatively, the compound decrease alters CaaX prenyl protease expression or activity. The compound 3o can be, e.g., (i) a CaaX prenyl protease polypeptide; (ii) a nucleic acid encoding a CaaX
prenyl protease polypeptide; (iii) a nucleic acid that increases expression of a nucleic acid that encodes a CaaX prenyl protease polypeptide ; (iv) a nucleic acid that decreases the expression of a nucleic acid that encodes a CaaX prenyl protease polypeptide; (v) a CaaX prenyl protease antisense nucleic acid and derivatives, fragments, analogs and homologs thereof. A nucleic acid that increases expression of a nucleic acid that encodes a CaaX prenyl protease polypeptide includes, e.g., promoters, enhancers. The nucleic acid can be either endogenous or exogenous. Preferably, the compound is a CaaX
prenyl protease polypeptide or a nucleic acid encoding a CaaX prenyl protease polypeptide. For example the compound comprises the nucleic acid sequence of SEQ ID NO:1, 14, or 17 or fragement thereof. Alternatively, the compound is a CaaX prenyl protease antisence nucleic acid. For example the compound comprises the nucleic acid sequence of SEQ ID
NO: 16, 19 or 20.
I o In various aspects the transgenic plant has an altered phenotype as compared to a wild type plant (i.e., untransformed). By altered phenotype is meant that the plant has a one or more characteristic that is different from the wild type plant. For example, the transgenic plant has an increased resistence to stress. Increased stress resistance is meant that the transgenic plant can grow under stress conditions (e.g., high salt, decreased water, low temperatures, high temperatures) or under conditions that normally inhibit the growth of an untransformed Stresses include, for example, chilling stress, heat stress, heat shock, salt stress, water stress (i. e, drought), nutritional stress, disease, grazing pests, wound healing, pathogens such as for example fungi, bacteria, nematodes, viruses or parasitic weed and herbicides. Methodologies to determine plant growth or response 2o to stress include for example, height measurements, weight or biomass measurements, leaf area or number, ability to flower, water use, transpiration rates and yield.
Alternatively, the transformed plant has an increased (i.e., enhanced) ABA
sensitivity.
The enhanced ABA sensitivity is at the seedling growth stage. Alternatively, the enhanced ABA sensitivity is at the mature plant stage. Additional altered phenotypes include for example, enhanced vegetative growth (e.g., increased leaf number, thickness and overall biomass), delayed reproductive growth (e.g., flowering later);
enhanced seedling vigor (e.g.,increased root biomass and length), enhanced lateral root formation and therefore soil penetration more extensive vascular system resulting in an enhanced transport system.
3o The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Gossypium, Picea, Caco, and Populus.
Screening Methods The isolated nucleic acid molecules of the invention can be used to express CPP
protein (e.g., via a recombinant expression vector in a host cell), to detect CPP mRNA
(e.g., in a biological sample) or a genetic lesion in a CPP gene, and to modulate CPP
1 o activity, as described further, below. In addition, the CPP proteins can be used to screen compounds that modulate the CPP protein activity or expression. In addition, the anti-CPP antibodies of the invention can be used to detect and isolate CPP
proteins and modulate CPP activity.
The invention provides a method (also referred to herein as a "screening assay") 15 for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to CPP proteins or have a stimulatory or inhibitory effect on, e.g., CPP protein expression or CPP
protein activity.
The invention also includes compounds identified in the screening assays described herein.
20 In one embodiment, the invention provides assays for screening candidate or test compounds which bind to a CPP protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic 25 library methods requiring deconvolution; the "one-bead one-compound"
library method;
and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds.
See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.
3o A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U. S A. 91: 11422; Zuckermann, et al., 1994. J.
Med. Chem.
37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew.
Chem. Int.
Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:
2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No.
5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc.
Natl. Acad.
Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390;
Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad.
Sci. U.S.A.
87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No.
5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a CPP
protein, or a biologically-active portion thereof, is contacted with a test compound and 2o the ability of the test compound to bind to a CPP protein determined. The cell, for example, can be of mammalian origin, plant cell or a yeast cell. Determining the ability of the test compound to bind to the CPP protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the CPP protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ~25I, 3s5, laC, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a CPP
protein, or a biologically-active portion thereof, with a known compound which binds CPP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CPP protein, wherein determining the ability of the test compound to interact with a CPP protein comprises determining the ability of the test compound to preferentially bind to CPP protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a CPP protein, or a biologically-active portion thereof, with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the CPP protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the activity of CPP
or a biologically-active portion thereof can be accomplished, for example, by determining the to ability of the CPP protein to bind to or interact with a CPP target molecule. As used herein, a "target molecule" is a molecule with which a CPP protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a CPP
interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A CPP target molecule can be a non-CPP molecule or a CPP
protein or polypeptide of the invention In one embodiment, a CPP target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound molecule) through the cell membrane and into the cell. The target, for 2o example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with CPP.
Determining the ability of the CPP protein to bind to or interact with a CPP
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the CPP protein to bind to or interact with a CPP target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a 3o CPP-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a CPP protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the CPP
protein or biologically-active portion thereof. Binding of the test compound to the CPP
protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the CPP protein or biologically-active portion thereof with a known compound which binds CPP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CPP protein, wherein determining the ability of the test compound to interact with a CPP protein comprises determining the ability of the test compound to preferentially bind to CPP or biologically-active portion thereof as compared to the 1 o known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting CPP protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the CPP protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of CPP can be accomplished, for example, by determining the ability of the CPP protein to bind to a CPP target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of CPP
protein can be accomplished by determining the ability of the CPP protein further modulate a CPP
2o target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described above.
In yet another embodiment, the cell-free assay comprises contacting the CPP
protein or biologically-active portion thereof with a known compound which binds CPP
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CPP protein, wherein determining the ability of the test compound to interact with a CPP protein comprises determining the ability of the CPP protein to preferentially bind to or modulate the activity of a CPP target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of CPP protein. In the case of cell-free assays comprising the membrane-bound form of CPP protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of CPP protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton X-114, Thesit~, Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either CPP protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to CPP
protein, or interaction of CPP protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-CPP fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or CPP protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and 2o pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.
Alternatively, the complexes can be dissociated from the matrix, and the level of CPP protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the CPP protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated CPP protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation 3o kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with CPP protein or target molecules, but which do not interfere with binding of the CPP
protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or CPP protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CPP protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the CPP protein or target molecule.
In another embodiment, modulators of CPP protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of CPP mRNA or protein in the cell is determined. The level of expression of CPP
mRNA
or protein in the presence of the candidate compound is compared to the level of expression of CPP mRNA or protein in the absence of the candidate compound.
The to candidate compound can then be identified as a modulator of CPP mRNA or protein expression based upon this comparison. For example, when expression of CPP
mRNA
or protein is greater (i. e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of CPP mRNA or protein expression. Alternatively, when expression of CPP
15 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CPP mRNA or protein expression. The level of CPP mRNA or protein expression in the cells can be determined by methods described herein for detecting CPP mRNA or protein.
20 In yet another aspect of the invention, the CPP proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No.
5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J.
Biol. Chem.
268: 12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that 25 bind to or interact with CPP ("CPP-binding proteins" or "CPP-by") and modulate CPP
activity. Such CPP-binding proteins are also likely to be involved in the propagation of signals by the CPP proteins as, for example, upstream or downstream elements of the CPP pathway.
The two-hybrid system is based on the modular nature of most transcription 3o factors, which consist of separable DNA-binding and activation domains.
Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for CPP is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a CPP-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor.
Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with CPP.
In yet another aspect of the invention are methods which utilize the transgenic plants of the invention to identify CPP-interacting components via genetic screening protocols. These components can be for example, regulatory elements which modify CPP-gene expression, interacting proteins which directly modify CPP activity or interacting proteins which modify components of the same signal transduction pathway and thereby exert an effect on the expression or activity of CPP. Briefly, genetic screening protocols are applied to the transgenic plants of the invention and in so doing identify related genes which are not identified using a wild type background for the screen. For example an activation tagged library (Weigel, et al., 2000. Plant Physiol.
122: 1003-1013), can be produced using the transgenic plants of the invention as the genetic background. Plants are then screened for altered phenotypes from that displayed 2o by the parent plants. Alternative methods of generating libraries from the transgenic plants of the invention can be used, for example, chemical or irradiation induced mutations, insertional inactivation or insertional activation methods.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof.
EXAMPLES
Example 1: RT-PCR amplification and cloning of CaaX prenyl proteases Total RNA was isolated from leaf tissue ofArabidopsis thaliana, Brassica napus and Glycine max, using the Qiagen RNeasy kit and used as template to amplify the CPP
genes by RT-PCR. Reaction conditions were as follows; 1X reaction buffer (IOmM
Tris HCI pH 8.8, l.SmM MgCl2, SOmM KCl), dNTP's at 200~M, 1pM AtCPP BamFW and AtCPP SmaRV primers, 2.5U. Pfu DNA polymerase, and template plus water to a final volume of 100pL. Reactions were run at 1 minute 94°C, 1 minute 60°C, 1 minute 72°C, for 30 cycles. Primers used to PCR amplify Arabidopsis and Brassica sequences were those identified by SEQ ID N0:6 and SEQ ID N0:7. Primers used to PCR amplify the Glycine sequence were those identified by SEQ ID N0:54 and SEQ ID N0:55. PCR
products were separated from the RT-PCR reaction mixture using the Qiagen PCR
column spin kit and ligated into the prepared cloning vector, pBluescript KS+.
The vector had been prepared by digestion with EcoRV and treated with Tag polymerase in the presence of dTTP to produce a 3' overhand suitable for ligation with the PCR
1o products. The ligation products were transformed into E. coli DHSa cells, positive colonies selected and the resulting inserts sequenced. The above methodology is applicable to obtain homologous sequences and may require alternative primers.
Table 1.

AtCPP BamFW: 5'-AAAGGATCCATGGCGATTCCTTTCATGG-3' (SEQ ID N0:6) AtCPP SmaRV: 5'-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3' (SEQ ID N0:7) GmCPP SmaFW: 5'-AAACCCGGGATGGCGTTTCCCTACATGGAAGCC -3' (SEQ ID N0:54) GmCPP SacRV: 5'-AAAGAGCTCTTAGTCTTCCTTCTTATCCGGTTCG -3' (SEQ ID N0:55) Example 2: Vector Construction Construction of the pBI121-AtCPP construct (SEQ ID NO: 4) was prepared as follows. The pBI121 vector was digested with BamHI and SmaI. The AtCPP, 1.4 kb DNA fragment from RT-PCR (SEQ ID NO: 1) was digested with BamHI and SmaI and ligated into the pBI121 vector. The GUS sequence was then removed by digestion with SmaI and EcoICRI and the vector ligated after purification of the vector from the GUS
insert to produce the pBI121-AtCPP vector (Figure 1A). This construct was used to further generate constructs expressing the CPP gene from Brassica and Glycine.
To produce the pBI121-BnCPP construct (SEQ ID N0:47) primer pairs identified by SEQ
ID N0:6 and SEQ ID N0:7 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-GmCPP construct (SEQ

ID N0:41 ) primer pairs identified by SEQ ID N0:54 and SEQ ID NO:55 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.
Construction of the pBI121-antisense-AtCPP construct (SEQ ID N0:35). The antisense fragment was produced using PCR amplification with SEQ ID NO:1 as template and primers identified as SEQ ID NO:11 and SEQ ID N0:12, listed in Table 2.
This fragment was digested with BamHI and SmaI and used to replace the sense fragment of the pBI121-AtCPP construct (SEQ ID NO: 4), to yield SEQ ID N0:35 (Figure 1B) . This construct, SEQ ID N0:35, was used to further generate constructs expressing the antisense CPP gene from Brassica and Glycine. To produce the pBIl21-1o antisense-BnCPP construct (SEQ ID N0:49) primer pairs identified by SEQ ID
N0:56 and SEQ ID N0:57 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-antisense-GmCPP
construct (SEQ ID N0:43) primer pairs identified by SEQ ID N0:58 and SEQ ID N0:59 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.
Construction of the pBI121-HP-AtCPP construct (SEQ ID NO: 5). The cloning strategy involved truncating the GUS gene of pBI l 21 and flanking the GUS
sequence with a AtCPP fragment in the antisense orientation upstream of the GUS and in the sense orientation on the downstream side of GUS. The pBI121 vector was digested with SmaI
and SacI, the GUS sequence and the vector fragments were purified from one another.
2o The isolated GUS fragment was digested using EcoRV and the 1079 bp. blunt ended EcoRVlSacI fragment isolated. This was ligated back into the digested parent vector at the SmaIlSacI sites. This intermediate vector was used in the subsequent production of the hair-pin vectors. The AtCPP fragment to be used as the gene specific hair-pin sequence was isolated by PCR. Primers identified as SEQ ID N0:8 and SEQ ID
N0:9, listed in Table 2, were used to generate a 596 by fragment. Cloning of the sense orientation fragment was achieved by digesting the PCR AtCPP fragment with SacI and ligation into the SacI site at the 3' end of GUS. To insert the same fragment upsteam of GUS, the BamHI site was opened and the ends blunted with Klenow. The PCR
amplified AtCPP fragment was digested with EcoICRI, which is an isoschizomer of SacI but leaves 3o blunt ends, and ligated into the blunted BamHI site of the vector to yield the final construct (Figure 1 C). The intermediate construct used to produce SEQ ID NO:S
above contained only the truncated GUS gene and no CPP sequences this intermediate vector was used to further generate constructs expressing hair-pin CPP gene constructs from Brassica and Glycine. To produce the pBI121-HP-BnCPP construct (SEQ ID N0:48) primer pairs identified by SEQ ID N0:58 and SEQ ID N0:59 are used to PCR
amplify the sense fragment and primer pairs identified by SEQ ID N0:60 and SEQ ID
N0:61 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above. To produce the pBIl21-HP-GmCPP
construct (SEQ ID N0:42) primer pairs identified by SEQ ID N0:62 and SEQ ID
N0:63 are used to PCR amplify the sense fragment and primer pairs identified by SEQ
ID
N0:64 and SEQ ID N0:65 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above.
to The above vector constructs were modified to place the genes under the control of alternative promoters, such as, but not limited to, the RD29A or MuA . This was accomplished by excising the 35S promoter sequence and replacing it with an appropriate promoter sequence. In this way SEQ ID NO's:39 and 40 were generated and SEQ ID NO's:38, 41-53 can be constructed.
Table 2 AtCPP-HP-SacFW 5'-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3' (SEQ ID N0:8) AtCPP-HP-SacRV 5'-ATTGAGCTCCCAATGTCCAAGCTCGTGTGCAATA-3' (SEQ ID N0:9) 2o AtCPP-anti-SmaFW 5'-AAACCCGGGATGGCGATTCCTTTCATGG-3' (SEQ ID NO:11) AtCPP-anti-BamRV 5'-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCCA-3' (SEQ ID N0:12) BnCPP-anti-SmaFW 5'-AAACCCGGGATGGCGATTCCTTTCATGG -3' (SEQ ID N0:56) BnCPP-anti-BamRV 5'-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCC -3' (SEQ ID N0:57) 3o BnCPP-HP-Sac-FW 5'- AAAGAGCTCTTCTACCAATGGTGGGACTCG -3' (SEQ ID N0:58) BnCPP-HP-Sac-RV 5'- AAAGAGCTCCCAGTGTCCCAGCTCGTGTG -3' (SEQ ID N0:59) BnCPP-HP-BamFW 5'- AAAGGATCCTTCTACCAATGGTGGGACTCG -3' (SEQ ID N0:60) BnCPP-HP-XbaRV 5'- AAATCTAGACCAGTGTCCCAGCTCGTGTG -3' (SEQ ID N0:61) GmCPP-HP-Sac-FW
5'-GATGAGCTCACAAGATCAAGTCACAGCAATGCCT-3' (SEQ ID N0:62) GmCPP-HP-Sac-RV 5'- AAAGAGCTCCCGGTTCGTCCAGCGCGGCC -3' (SEQ ID N0:63) GmCPP-HP-BamFW
5'-GATGGATCCACAAGATCAAGTCACAGCAATGCCT-3' (SEQ ID N0:64) GmCPP-HP-XbaRV 5'- CCTTCTAGACCGGTTCGTCCAGCGCGGCC -3' (SEQ ID N0:65) 1 o Example 3: Sequence Analysis Arabidopsis thaliana CPP (AtCPP) A disclosed nucleic acid of 1275 nucleotides (SEQ ID NO:l) and also referred to as AtCPP, is shown in Table 3.
Table 3A. AtCPP Nucleotide Sequence (SEQ ID NO:1).
ATGGCGATTCCTTTCATGGAAACCGTCGTGGGTTTTATGATAGTGATGTACATTTTTGAG
ACGTATTTGGATCTGAGGCAACTCACTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTG
GTTGGTGTAATTAGCCAAGAGAAGTTTGAGAAATCACGAGCATACAGTCTTGACAAAAGC
TATTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTT
GGGATCTTGCCTTGGTTTTGGAAGATGTCTGGAGCTGTTTTACCGAGGTTGGGCCTTGAT
CCGGAGAATGAAATACTGCATACTCTTTCATTCTTGGCTGGTGTTATGACATGGTCACAG
ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
AACAAACAAACAATATGGATGTTCATTAGGGACATGATCAAAGGAACATTCCTCTCTGTC
ATACTAGGCCCACCCATTGTTGCTGCGATAATTTTCATAGTCCAGAAAGGAGGTCCTTAT
CTTGCCATCTATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATAC
CCGGTCTTGATAGCACCGCTCTTCAACAAATTCACTCCTCTTCCAGATGGAGACCTCCGG
GAGAAGATTGAGAAACTTGCTTCTTCCCTAAAGTTTCCTTTGAAGAAGCTGTTTGTTGTC
GATGGATCTACAAGGTCAAGCCATAGCAATGCTTACATGTATGGTTTCTTTAAGAACAAA
AGGATTGTTCTTTATGATACGTTGATTCAGCAGTGCAAGAATGAGGATGAAATTGTGGCG
GTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACTCGTTCATTGCA
GTTCAAATCCTTGCCTTCTTACAATTTGGAGGATACACTCTTCTCAGAAACTCCACTGAT
CTCTTCAGGAGTTTCGGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG
CACACTGTAATACCACTGCAACATCTAGTAAGCTTTGGCCTGAACCTCGTTAGTCGAGCG
TTTGAGTTTCAGGCTGATGCTTTTGCTGTGAAGCTTGACTATGCAAAAGATCTTCGTCCT
GCTCTAGTGAAACTACAGGAAGAGAACTTATCAACAATGAACACTGATCCATTGTACTCA
GCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGCTTCGAGCCACTGATGGAGAAGAC
AAGAAGACAGATTAA

A disclosed CPP polypeptide (SEQ ID N0:2) encoded by SEQ ID NO:1 has 424 amino acid residues and is presented in Table 3B using the one-letter amino acid code.
Table 3B. Encoded CPP protein sequence (SEQ ID N0:2).
MAIPFMETVVGFMIVMYIFETYLDLRQLTALKLPTLPKTLVGVISQEKFEKSRAYSLDKS
YFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLAGVMTWSQ
ITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFIVQKGGPY
LAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFPLKKLFVV
DGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKNEDEIVAVIAHELGHWKLNHTTYSFIA
VQILAFLQFGGYTLLRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFGLNLVSRA
FEFQADAFAVKLDYAKDLRPALVKLQEENLSTMNTDPLYSAYHYSHPPLVERLRATDGED
KKTD
The present invention also includes a nucleic acid sequence complimentary to the Arabidopsis thaliana CaaX prenyl protease of SEQ ID NO:l. The disclosed complimentary sequence is shown as SEQ ID N0:20.
SEQ ID N0:20 TTAATCTGTCTTCTTGTCTTCTCCATCAGTGGCTCGAAGCCTTTCAACAAGAGGAGGAT
GTGAGTAGTGATAAGCTGAGTACAATGGATCAGTGTTCATTGTTGATAAGTTCTCTTCC
TGTAGTTTCACTAGAGCAGGACGAAGATCTTTTGCATAGTCAAGCTTCACAGCAAAAGC
ATCAGCCTGAAACTCAAACGCTCGACTAACGAGGTTCAGGCCAAAGCTTACTAGATGTT
GCAGTGGTATTACAGTGTGCTGAAATATGATCAAACCAATGAGAACAGGCTGTGTATCA
AATCCGAAACTCCTGAAGAGATCAGTGGAGTTTCTGAGAAGAGTGTATCCTCCAAATTG
TAAGAAGGCAAGGATTTGAACTGCAATGAACGAGTATGTAGTGTGATTCAGTTTCCAAT
GTCCAAGCTCGTGTGCAATAACCGCCACAATTTCATCCTCATTCTTGCACTGCTGAATC
AACGTATCATAAAGAACAATCCTTTTGTTCTTAAAGAAACCATACATGTAAGCATTGCT
ATGGCTTGACCTTGTAGATCCATCGACAACAAACAGCTTCTTCAAAGGAAACTTTAGGG
AAGAAGCAAGTTTCTCAATCTTCTCCCGGAGGTCTCCATCTGGAAGAGGAGTGAATTTG
TTGAAGAGCGGTGCTATCAAGACCGGGTATATAGTCATCATCACTAGAGACAGGATAAA
CATGAATGCCCACAGATAGATGGCAAGATAAGGACCTCCTTTCTGGACTATGAAAATTA
TCGCAGCAACAATGGGTGGGCCTAGTATGACAGAGAGGAATGTTCCTTTGATCATGTCC
CTAATGAACATCCATATTGTTTGTTTGTTGAACCCATGCCGAGACTCGATCACGAAAGT
TGAGTACAAAGAAAATGGCAAATCAGTGATCTGTGACCATGTCATAACACCAGCCAAGA
ATGAAAGAGTATGCAGTATTTCATTCTCCGGATCAAGGCCCAACCTCGGTAAAACAGCT
CCAGACATCTTCCAAAACCAAGGCAAGATCCCAAAGAACAAAATTGCAGAGTCCATAAG

TATAGTTACAAACTCATGAACAAAGTGAAAATAGCTTTTGTCAAGACTGTATGCTCGTG
ATTTCTCAAACTTCTCTTGGCTAATTACACCAACCAAGGTTTTCGGGAGAGTTGGAAGC
TTGAGAGCAGTGAGTTGCCTCAGATCCAAATACGTCTCAAAAATGTACATCACTATCAT
AAAACCCACGACGGTTTCCATGAAAGGAATCGCCAT
Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5' and 3' non-translated regions. The percent identities of the Arabidopsis thaliana nucleotide sequence and its encoded amino acid sequence to that of other CPP sequences as determined by ClustalW analysis are shown in Figure 2.
l0 Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
Brassica napus CPP (BnCPP) 15 A disclosed nucleic acid of 1275 nucleotides (SEQ ID N0:14) and also referred to as BnCPP, is shown in Table 4.
Table 4A. BnCPP Nucleotide Sequence (SEQ ID N0:14).
ATGGCGATTCCTTTCATGGAAACCGTCGTTGGTTTTATGATAGTGATGTACGTTTTTGAGACGTA
TTTGGATCTGAGGCAACATACTGCTCTCAAGCTTCCCACTCTCCCAAAGACTTTGGTTGGAGTCA
TTAGCCAAGAGAAGTTTGAGAAATCTCGAGCTTACAGTCTTGACAAAAGCCATTTTCACTTTGTT
CATGAGTTTGTTACTATACTTATGGACTCTGCGATTCTGTTCTTTGGGATCTTGCCTTGGTTTTG
GAAGATATCTGGCGGCTTTCTACCAATGGTGGGACTCGATCCAGAGAATGAAATCCTGCACACTC
TTTCATTCTTGGCTGGTCTTATGACATGGTCACAGATCACTGATTTGCCATTTTCTTTGTACTCA
ACTTTCGTGATCGAGTCTCGGCATGGGTTCAACAAACAAACAATATGGATGTTCATTAGGGACAT
GATCAAAGGAATACTCCTCTCTGTCATACCTGCCCCTCCTATCGTTGCCGCAATTATTGTTATAG
TTCAGAAAGGAGGTCCTTACCTCGCCATCTATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTG
ATGATGACTATATACCCTGTTTTGATTGCACCTCTTTTCAACAAGTTCACTCCTCTTCCTGATGG
AGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTTTCCTCTGAAGAAGCTGTTTG
TTGTCGATGGATCTACAAGGTCAAGCCATAGTAATGCTTACATGTATGGTTTCTTCAAGAACAAA
AGGATTGTTCTTTATGACACATTGATTCAGCAGTGCCAGAATGAGAATGAAATTGTGGCGGTTAT
TGCACACGAGCTGGGACACTGGAAGCTGAATCACACTACATACTCGTTCATTGCTGTTCAAATCC
TTGCCTTCTTGCAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTT
GGTTTTGATACACAACCAGTTCTCATTGGTTTGATCATATTTCAGCACACTGTAATACCACTTCA
ACACCTAGTAAGCTTTGACCTCAACCTTGTTAGTCGAGCGTTTGAGTTTCAGGCTGATGCTTTTG
CAGTGAATCTTGGTTATGCAAAGGATCTACGTCCTGCCCTAGTGAAGCTACAGGAAGAGAACTTA

TCAGCGATGAACACAGACCCATTGTACTCAGCTTATCACTACTCACACCCTCCTCTTGTAGAGAG
GCTTCGAGCCATTGATGGAGAAGACAAGAAGACAGATTAA
A disclosed CPP polypeptide (SEQ ID NO:15) encoded by SEQ ID N0:14 has 424 amino acid residues and is presented in Table 4B using the one-letter amino acid code.
Table 4B. Encoded CPP protein sequence (SEQ ID NO:15).
NIAIPFMETVVGFMIVMYVFETYLDLRQHTALKLPTLPKTLVGVISQEKFEKSRAYSLDKSHFHF
VHEFVTILMDSAILFFGILPWFWKISGGFLPMVGLDPENEILHTLSFLAGLMTWSQITDLPFSL
YSTFVIESRHGFNKQTIWMFIRDMIKGILLSVIPAPPIVAAIIVIVQKGGPYLAIYLWAFMFIL
SLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFPLKKLFVVDGSTRSSHSNAYMYGF
FKNKRIVLYDTLIQQCQNENEIVAVIAHELGHWKLNHTTYSFIAVQILAFLQFGGYTLVRNSTD
LFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFDLNLVSRAFEFQADAFAVNLGYAKDLRPALVK
LQEENLSAMNTDPLYSAYHYSHPPLVERLRAIDGEDKKTD
The present invention also includes a nucleic acid sequence complimentary to the Brassica napus CaaX prenyl protease of SEQ ID N0:14. The disclosed complimentary sequence is shown as SEQ ID N0:16.
l0 SEQ ID N0:16 TTAATCTGTCTTCTTGTCTTCTCCATCAATGGCTCGAAGCCTCTCTACAAGAGGAGGGT
GTGAGTAGTGATAAGCTGAGTACAATGGGTCTGTGTTCATCGCTGATAAGTTCTCTTCC
TGTAGCTTCACTAGGGCAGGACGTAGATCCTTTGCATAACCAAGATTCACTGCAAAAGC
ATCAGCCTGAAACTCAAACGCTCGACTAACAAGGTTGAGGTCAAAGCTTACTAGGTGTT

AAACCAAAACTCCTGAAGAGATCAGTGGAGTTTCTGACAAGAGTGTATCCTCCAAATTG
CAAGAAGGCAAGGATTTGAACAGCAATGAACGAGTATGTAGTGTGATTCAGCTTCCAGT
GTCCCAGCTCGTGTGCAATAACCGCCACAATTTCATTCTCATTCTGGCACTGCTGAATC
AATGTGTCATAAAGAACAATCCTTTTGTTCTTGAAGAAACCATACATGTAAGCATTACT

AAGAAGCAAGTTTCTCAATCTTCTCCCGGAGGTCTCCATCAGGAAGAGGAGTGAACTTG
TTGAAAAGAGGTGCAATCAAAACAGGGTATATAGTCATCATCACTAGAGACAGGATAAA
CATGAATGCCCACAGATAGATGGCGAGGTAAGGACCTCCTTTCTGAACTATAACAATAA
TTGCGGCAACGATAGGAGGGGCAGGTATGACAGAGAGGAGTATTCCTTTGATCATGTCC

TGAGTACAAAGAAAATGGCAAATCAGTGATCTGTGACCATGTCATAAGACCAGCCAAGA
ATGAAAGAGTGTGCAGGATTTCATTCTCTGGATCGAGTCCCACCATTGGTAGAAAGCCG
CCAGATATCTTCCAAAACCAAGGCAAGATCCCAAAGAACAGAATCGCAGAGTCCATAAG
TATAGTAACAAACTCATGAACAAAGTGAAAATGGCTTTTGTCAAGACTGTAAGCTCGAG
ATTTCTCAAACTTCTCTTGGCTAATGACTCCAACCAAAGTCTTTGGGAGAGTGGGAAGC
TTGAGAGCAGTATGTTGCCTCAGATCCAAATACGTCTCAAAAACGTACATCACTATCAT
AAAACCAACGACGGTTTCCATGAAAGGAATCGCCAT
Due to the nature of the cloning strategy the sequence presented is not full length to but is missing the 5' and 3' non-translated regions. The percent identities of the Brassica napus nucleotide sequence and its encoded amino acid sequence to that of other CPP
sequences as determined by ClustalW analysis are shown in Figure 2.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid 15 amplification of cDNA ends (RACE) technology or other such PCR techniques.
Glycine max CPP (GmCPP) A disclosed nucleic acid of 1275 nucleotides (SEQ ID N0:17) and also referred to as GmCPP, is shown in Table 5.
Table SA. GmCPP Nucleotide Sequence (SEQ ID N0:17).
ATGGCGTTTCCCTACATGGAAGCCGTTGTCGGATTTATGATATTAATGTACATTTTTGAAACTTA
CTTGGATGTGCGACAACATAGGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAGGGTGTTA
TCAGCCAAGAGAAATTTGAGAAATCTAGAGCCTATAGTCTTGATAAAAGCCACTTCCATTTTGTT
CACGAGTTTGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGGGGTATTGCCCTGGTTTTG
GAAGAAATCAGGAGATTTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCATACCC
TTGCCTTCTTAGCAGGGCTGATGATTTGGTCACAGATAACAGATTTGCCCTTTTCTCTGTACTCA
ACTTTTGTGATTGAGGCCCGTCATGGTTTTAATAAGCAAACACCATGGTTATTCTTTAGGGACAT
GCTTAAAGGAATTTTCCTTTCTGTAATAATTGGTCCACCTATTGTGGCTGCAATCATTGTAATAG
TACAGAAAGGAGGTCCATACTTGGCCATCTATCTTTGGGTTTTTACGTTTGGTCTTTCTATTGTG
ATGATGACCCTTTATCCAGTACTAATAGCTCCACTCTTCAATAAGTTCACTCCACTTCCAGATGG
TCAACTCAGGGAGAAAATCGAGAAACTTGCTTCCTCCCTCAACTATCCGTTAAAGAAACTATTTG
TTGTCGATGGATCCACAAGATCAAGTCACAGCAATGCCTATATGTATGGATTCTTCAAGAACAAG
AGGATTGTCCCTTATGACACATTAATTCAACAGTGCAAAGACGATGAGGAAATTGTTGCTGTTAT
TGCCCATGAGTTGGGACACTGGAAGCTCAACCATACTGTGTACACATTTGTTGCTATGCAGATTC
TTACACTTCTACAATTTGGAGGATATACACTAGTGCGAAATTCAGCTGATCTGTATCGAAGCTTT

GGGTTTGATACGCAGCCAGTCCTCATTGGGCTCATCATATTTCAGCATACTGTAATCCCACTTCA
GCAATTGGTCAGCTTTGGTCTGAACCTAGTCAGCCGATCATTTGAATTTCAGGCTGATGGCTTTG
CCAAGAAGCTTGGATATGCATCTGGATTACGCGGTGGTCTTGTGAAACTACAGGAGGAGAATCTG
TCAGCTATGAATACAGATCCTTGGTACTCTGCTTATCACTATTCTCATCCTCCCCTTGTTGAAAG
ATTGGCCGCGCTGGACGAACCGGATAAGAAGGAAGACTAA
A disclosed CPP polypeptide (SEQ ID N0:18) encoded by SEQ ID N0:17 has 424 amino acid residues and is presented in Table SB using the one-letter amino acid code.
Table SB. Encoded CPP protein sequence (SEQ ID N0:18).
MAFPYMEAVVGFMILMYIFETYLDVRQHRALKLPTLPKTLEGVISQEKFEKSRAYSLDKS
HFHFVHEFVTIVTDSTILYFGVLPWFWKKSGDFMTIAGFNAENEILHTLAFLAGLMIWSQ
ITDLPFSLYSTFVIEARHGFNKQTPWLFFRDMLKGIFLSVIIGPPIVAAIIVIVQKGGPY
LAIYLWVFTFGLSIVMMTLYPVLIAPLFNKFTPLPDGQLREKIEKLASSLNYPLKKLFVV
DGSTRSSHSNAYMYGFFKNKRIVPYDTLIQQCKDDEEIVAVIAHELGHWKLNHTVYTFVA
MQILTLLQFGGYTLVRNSADLYRSFGFDTQPVLIGLIIFQHTVIPLQQLVSFGLNLVSRS
FEFQADGFAKKLGYASGLRGGLVKLQEENLSAMNTDPWYSAYHYSHPPLVERLAALDEPD
KKED
The present invention also includes a nucleic acid sequence complimentary to the Glycine max CaaX prenyl protease of SEQ ID N0:17. The disclosed complimentary sequence is shown as SEQ ID N0:19.
to SEQ ID N0:19 TTAGTCTTCCTTCTTATCCGGTTCGTCCAGCGCGGCCAATCTTTCAACAAGGGGAGGAT
GAGAATAGTGATAAGCAGAGTACCAAGGATCTGTATTCATAGCTGACAGATTCTCCTCC
TGTAGTTTCACAAGACCACCGCGTAATCCAGATGCATATCCAAGCTTCTTGGCAAAGCC
ATCAGCCTGAAATTCAAATGATCGGCTGACTAGGTTCAGACCAAAGCTGACCAATTGCT

AACCCAAAGCTTCGATACAGATCAGCTGAATTTCGCACTAGTGTATATCCTCCAAATTG
TAGAAGTGTAAGAATCTGCATAGCAACAAATGTGTACACAGTATGGTTGAGCTTCCAGT
GTCCCAACTCATGGGCAATAACAGCAACAATTTCCTCATCGTCTTTGCACTGTTGAATT
AATGTGTCATAAGGGACAATCCTCTTGTTCTTGAAGAATCCATACATATAGGCATTGCT

AGGAAGCAAGTTTCTCGATTTTCTCCCTGAGTTGACCATCTGGAAGTGGAGTGAACTTA
TTGAAGAGTGGAGCTATTAGTACTGGATAAAGGGTCATCATCACAATAGAAAGACCAAA

CGTAAAAACCCAAAGATAGATGGCCAAGTATGGACCTCCTTTCTGTACTATTACAATGA
TTGCAGCCACAATAGGTGGACCAATTATTACAGAAAGGAAAATTCCTTTAAGCATGTCC
CTAAAGAATAACCATGGTGTTTGCTTATTAAAACCATGACGGGCCTCAATCACAAAAGT
TGAGTACAGAGAAAAGGGCAAATCTGTTATCTGTGACCAAATCATCAGCCCTGCTAAGA
AGGCAAGGGTATGCAGTATTTCATTCTCAGCATTGAAACCAGCTATTGTCATAAAATCT
CCTGATTTCTTCCAAAACCAGGGCAATACCCCAAAGTACAAAATTGTAGAGTCTGTCAC
TATTGTCACAAACTCGTGAACAAAATGGAAGTGGCTTTTATCAAGACTATAGGCTCTAG
ATTTCTCAAATTTCTCTTGGCTGATAACACCCTCTAAAGTCTTTGGAAGAGTAGGAAGT
TTGAGGGCCCTATGTTGTCGCACATCCAAGTAAGTTTCAAAAATGTACATTAATATCAT
AAATCCGACAACGGCTTCCATGTAGGGAAACGCCAT
Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5' and 3' non-translated regions. The percent identities of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other CPP
IS sequences as determined by ClustalW analysis are shown in Figure 2.
Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.
The CPP nucleic acids and amino acids disclosed above have homology to other disclosed CPP sequences (GenBank ID NOs: AL161491 (AT4g01320), AF007269 and AF353722; WO 02/16625 A2 ). The homology between these and other sequences is shown in the ClustalW alignment analysis shown in Tables 6A-6B.
Table 6A. ClustalW Nucleic Acid Analysis of CaaX Prenyl Protease 1: PPI-AtCPP SEQ ID NO:1 2: PPI-BnCPP SEQ ID N0:14 3: PPI-GmCPP SEQ ID N0:17 4: BASF AT1 SEQ ID N0:21 5: BASF SEQ ID N0:23 6: BASF-Corn SEQ ID N0:25 7: BASF-Gm SEQ ID N0:27 8: AFC 1 SEQ ID N0:29 9: AT4g01320 SEQ ID N0:31 3s 10: AF007269SEQ ID N0:33 CLUSTAL W (1.81) multiple sequence alignment PPI-GmCPP ______________________________________---___________________ BASF-Gm _____________________________________--_____________________ AT4g01320 ____________________________________________________________ PPI-AtCPP ________________________________-___________________________ BASF_AT2 __________________________-__________________-____________-_ afcl _______________-__________________-___-_____________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ BASF-Corn -___________________________________________________________ PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ 1S AT4g01320 ____________________________________________________________ PPI-AtCPP _____________________________________-______________________ BASF_AT2 ______________________________________________-_____________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP _____________________-____-_________________________________ BASF-Corn ______________________________________-_____________________ 2S PPI-GmCPP
BASF-Gm ____________________________________________________________ AT4g01320 ________________________________-___________________________ PPI-AtCPP _____________________________________________-____-_________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ___________________-________________________________________ PPI-BnCPP ____________________________________________________________ BASF-Corn ____________________________________________________________ PPI-GmCPP ____________________________________________________________ BASF-Gm _______________--___________________________________________ AT4g01320 __-___________________-_____________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl _-_______________-_____________________-____________________ BASF_AT1 ____________________________________________________________ 4S PPI-BnCPP _____________________________________-_____________________-BASF-Corn ____________________________________________-_____-_________ SO PPI-GmCPP
BASF-Gm ____________________________________________-_____-_________ AT4g01320 ________-___________________________________________________ PPI-AtCPP ____________________________________________-_______________ SS BASF_AT2 ____________________________________________________________ afcl ___________________________________-__-_____-_______________ BASF_AT1 ___-________________________________________________________ PPI-BnCPP ______________________________-____-__-_____________________ BASF-Corn ____________________________________________-_______________ PPI-GmCPP ________________-_-___-_________________-____-____________-_ BASF-Gm ___________________________________-________-_______________ AT4g01320 _____-__________________________________-_____-___________-_ PPI-AtCPP ______________________-__-__________________________________ BASF_AT2 __-__-_________-__________________________________-________-afcl ____________________________________________--_____________-BASF_AT1 ____________________________________________________________ PPI-BnCPP _____________________-______________________________________ BASF-Corn ____________________________________________________________ S
PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ 1O PPI-AtCPP _______________________________________________________-____ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ 1S BASF-Corn ____________________________________________________________ PPI-GmCPP ____________________________________________________________ BASF-Gm --------------------------------------CTAATACGACTCACTATAGGGC
20 AT4g01320 ____________________________________________________________ PPI-AtCPP -___________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ 2S BASF_AT1 ____________________________________________________________ PPI-BnCPP _________________________________________________________-__ BASF-Corn ____________________________________________________________ PPI-GmCPP ____________________________________________________________ 30 BASF-Gm AAGCAGTGGTAACAACGCAGAGTACGCGGGGGGAGACGCATGGTTCTGAACTAATTGTTA
AT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ 3S afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ BASF-Corn ____________________________________________________________ PPI-GmCPP ____________________________________________________________ BASF-Gm TAAATAATACCTAAAATTTTGAGTTGTCCTAAACATTGGGGTTTAAACAAATCCAATCTC

AT4g01320 _______________________-____________________________________ 4SPPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ SOBASF-Corn ____________________________________________________________ PPI-GmCPP ____________________________________________________________ BASF-Gm TCAATATAAAACCCAATGATCTCACC--CTCACTCCGTTTCTGATTTCTCACTCTTCGTT

SSAT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF ____________________________________________________________ _ ____________________________________________________________ afcl 6OBASF_AT1 ____________________________________________________________ PPI-BnCPP __-_________________________________________________________ BASF-Corn ________________________________-___________________________ PPI-GmCPP ---------------------------------ATGGCGTTTCCC--TACATGGAAGCCG

6SBASF-Gm TCTCGTTCGGTTCATCAGCGTGTGTCTCAGC-CATGGCGTTTCCC--TACATGGAAGCCG

AT4g01320 ---------------------------------ATGGCGATTCCT--TTCATGGAAACCG

PPI-AtCPP ---------------------------------ATGGCGATTCCT--TTCATGGAAACCG

BASF ---------------------------------ATGGCGATTCCT--TTCATGGA~~v~~~

_ ---------------------------------ATGGCGATTCCT--TTCATGGAAACCG
afcl BASF ---------------------------------ATGGCGATTCCT--TTCATGGAAACCG

_ ---------------------------------ATGGCGATTCCT--TTCATGGAAACCG
PPI-BnCPP

S BASF-Corn ______________________________________________-_____________ PPI-GmCPP TTGTCGGATTTATGATATTAATGTACATTTTTGAAACTTACTTGGATGTGCGACAACATA

BASF-Gm TTGTCGGATTTATGATATTAATGTACATTTTTGAAACTTACTTGGATGTGCGACAACATA

AT4g01320 TCGTGGGTTTTATGATAGTGATGTACATTTTTGAGACGTATTTGGATCTGAGGCAACTCA

PPI-AtCPP TCGTGGGTTTTATGATAGTGATGTACATTTTTGAGACGTATTTGGATCTGAGGCAACTCA

BASF TCGTGGGTTTTATGATAGTGATGTACATTTTTGAGACGTATTTGGATCTGAGGCAACTCA

_ TCGTGGGTTTTATGATAGTGATGTACATTTTTGAGACGTATTTGGATCTGAGGCAACTCA
afcl BASF TCGTGGGTTTTATGATAGTGATGTACATTTTTGAGACGTATTTGGATCTGAGGCAACTCA

1S_ TCGTTGGTTTTATGATAGTGATGTACGTTTTTGAGACGTATTTGGATCTGAGGCAACATA
PPI-BnCPP

BASF-Corn __________________________________________________-_________ PPI-GmCPP GGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAGGGTGTTATCAGCCAAGAGAAAT

BASF-Gm GGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAAGGTGTTATCAGCCAAGAGAAAT

2OAT4g01320 CTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTGGTTGGTGTAATTAGCCAAGAGAAGT

PPI-AtCPP CTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTGGTTGGTGTAATTAGCCAAGAGAAGT

BASF CTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTGGTTGGTGTAATTAGCCAAGAGAAGT

_ CTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTGGTTGGTGTAATTAGCCAAGAGAAGT
afcl 2SBASF_AT1 CTGCTCTCAAGCTTCCAACTCTCCCGAAAACCTTGGTTGGTGTAATTAGCCAAGAGAAGT

PPI-BnCPP CTGCTCTCAAGCTTCCCACTCTCCCAAAGACTTTGGTTGGAGTCATTAGCCAAGAGAAGT

BASF-Corn _________-__________________________________________________ 30PPI-GmCPP TTGAGAAATCTAGAGCCTATAG--------------------------------------BASF-Gm TTGAGAAATCTAGAGCCTATAG--------------------------------------AT4g01320 TTGAGAAATCACGAGCATACAG--------------------------------------PPI-AtCPP TTGAGAAATCACGAGCATACAG--------------------------------------3SBASF_AT2 TTGAGAAATCACGAGCATACAG--------------------------------------afcl TTGAGAAATCACGAGCATACAG--------------------------------------BASF_AT1 TTGAGAAATCACGAGCATACAG--------------------------------------PPI-BnCPP TTGAGAAATCTCGAGCTTACAG--------------------------------------BASF-Corn ____________________________________________________________ PPI-GmCPP ___________________________________-______TCTTGATAAA---AGCCA

BASF-Gm ------------------------------------------TCTTGATAAA---AGCCA

AT4g01320 ------------------------GGATATCATCACTGAGAACTTTAATATATGCAGCTA

PPI-AtCPP _____________-____________________________TCTTGACAAA---AGCTA

BASF_AT2 ------------------------------------------TCTTGACAAA---AGCTA

afcl ------------------------------------------TCTTGACAAA---AGCTA

BASF ------------------------------------------TCTTGACAAA---AGCTA

SO_ __________________________________________TCTTGACAAA---AGCCA
PPI-BnCPP

BASF-Corn ____________________________________________________________ PPI-GmCPP CTTCCATTTTGTTCACGAGTTTGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGG

BASF-Gm CTTCCATTTTGTTCACGAGTTTGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGG

SSAT4g01320 TTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTTGG

PPI-AtCPP TTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTTGG

BASF_AT2 TTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTTGG

afcl TTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTTGG

60BASF_AT1 TTTTCACTTTGTTCATGAGTTTGTAACTATACTTATGGACTCTGCAATTTTGTTCTTTGG

PPI-BnCPP TTTTCACTTTGTTCATGAGTTTGTTACTATACTTATGGACTCTGCGATTCTGTTCTTTGG

BASF-Corn ____________________________________________________________ PPI-GmCPP GGTATTGCCCTGGTTTTGGAAG--------------------------------------6SBASF-Gm GGTATTGCCCTGGTTTTGGAAG--------------------------------------AT4g01320 GATCTTGCCTTGGTTTTGGAAG--------------------------------------PPI-AtCPP GATCTTGCCTTGGTTTTGGAAG--------------------------------------BASF_AT2 GATCTTGCCTTGGTTTTGGAAG--------------------------------------afcl GATCTTGCCTTGGTTTTGGAAG--------------------------------------BASF_AT1 GATCTTGCCTTGGTTTTGGAAG--------------------------------------PPI-BnCPP GATCTTGCCTTGGTTTTGGAAG--------------------------------------BASF-Corn ____________________________________________________________ PPI-GmCPP ______-_________________________________-_______~TCAGGAGAT

BASF-Gm ________________________________________________~TCAGGAGAT

AT4g01320 __-_____________________________________________ATGTCTGGAGCT

PPI-AtCPP ________________________________________________ATGTCTGGAGCT

BASF ________________________________________________ATGTCTGGAGCA

_ ________________________________________________ATGTCTGGAGCT
afcl BASF ______________________-_________________________ATGTCTGGAGCT

1S_ __________-_____________________________________ATATCTGGCGGC
PPI-BnCPP

BASF-Corn ____________________________________________________________ PPI-GmCPP TTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCATACCCTTGCCTTCTTA

BASF-Gm TTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCATACCCTTGCCTTCTTA

AT4g01320 GTTTTACCGAGGTTGGGCCTTGATCCAGAGAATGAAATACTGCATACTCTTTCATTCTTG

PPI-AtCPP GTTTTACCGAGGTTGGGCCTTGATCCGGAGAATGAAATACTGCATACTCTTTCATTCTTG

BASF GTTTTACCGAGGTTGGGCCTTGATCCAGAGAATGAAATACTGCATACTCTTTCATTCTTG

_ GTTTTACCGAGGTTGGGCCTTGATCCAGAGAATGAAATACTGCATACTCTTTCATTCTTG
afcl BASF GTTTTACCGAGGTTGGGCCTTGATCCAGAGAATGAAATACTGCATACTCTTTCATTCTTG

2S_ TTTCTACCAATGGTGGGACTCGATCCAGAGAATGAAATCCTGCACACTCTTTCATTCTTG
PPI-BnCPP

BASF-Corn ------------ACGAGGCTGAGTGCTGAGAATGAGATAATACACACCCTTGCTTTCTTA

* * * * ******** ** * ** ** *** * *****

PPI-GmCPP GCAGGGCTGATGATTTGGTCACAG------------------------------------BASF-Gm GCAGGGCTGATGATTTGGTCACAG------------------------------------3OAT4g01320 GCTGGTGTTATGACATGGTCACAG------------------------------------PPI-AtCPP GCTGGTGTTATGACATGGTCACAG------------------------------------BASF_AT2 GCTGGTGTTATGACATGGTCACAG------------------------------------afcl GCTGGTGTTATGACATGGTCACAG------------------------------------3SBASF_AT1 GCTGGTGTTATGACATGGTCACAC------------------------------------PPI-BnCPP GCTGGTCTTATGACATGGTCACAG------------------------------------BASF-Corn GCTGGTTCCATGGTTTGGTCGCAG------------------------------------** ** *** ***** **

4O PPI-GmCPP
BASF-Gm ____________________________________________________________ AT9g01320 ____________________________________________________________ PPI-AtCPP ______-_____________________________________________________ 4S BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________-_______________________________________________ BASF-Corn ____________________________________________________________ PPI-GmCPP ATAACAGATTTGCCCTTTTCTCTGTACTCAACTTTTGTGATTGAGGCCCGTCATGGTTTT
BASF-Gm ATAACAGATTTGCCCTTTTCTCTGTACTCAACTTTTGTGATTGAGGCCCGTCATGGTTTT
AT4g01320 ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC

SS PPI-AtCPP ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
BASF_AT2 ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
afcl ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
BASF_AT1 ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
PPI-BnCPP ATCACTGATTTGCCATTTTCTTTGTACTCAACTTTCGTGATCGAGTCTCGGCATGGGTTC
BASF-Corn ATTACAGACTTGCCGTTCTCTCTCTATTCAACTTTTGTTATAGAGGCTCGACATGGTTTT
** ** ** ***** ** *** * ** ******** ** ** *** * ** ***** **
PPI-GmCPP AATAAG-_-___________________________________________________ BASF-Gm AATAAG-_____________________________________________________ 6S AT4g01320 AACAAA-_____________________________________________________ PPI-AtCPP AACAAA--____________________________________-_______________ BASF AT2 AACAAA--____________________________________________________ afcl AACAAA-__________________________-__________________________ BASF_AT1 AACAAA-_____________________-________________________-______ PPI-BnCPP AACAAA-______-_____-________-____-___-_______________-______ BASF-Corn AACAAG-____________-________-_____-____-__________________-_ ** **

PPI-GmCPP --------------------------------CAAACACCATGGTTATTCTTTAGGGACA

BASF-Gm --------------------------------CAAACACCATGGTTATTCTTTAGGGACA

AT4g01320 --------------------------------CAAACAATATGGATGTTCATTAGGGACA

1OPPI-AtCPP --------------------------------CAAACAATATGGATGTTCATTAGGGACA

BASF --------------------------------CAAACAATATGGATGTTCATTAGGGACA

_ --------------------------------CAAACAATATGGATGTTCATTAGGGACA
afcl BASF --------------------------------CAAACAATATGGATGTTCATTAGGGACA

_ --------------------------------CAAACAATATGGATGTTCATTAGGGACA
PPI-BnCPP

15BASF-Corn --------------------------------CAAACTATATGGCTCTTCATTAGGGATA

***** **** * *** *******

PPI-GmCPP TGCTTAAAGGAATTTTCCTTTCTGTAATAATTGGTCCACCTATTGTGGCTGCAATCATTG

BASF-Gm TGCTTAAAGGAATTTTCCTTTCCGTAATAATTGGTCCACCTATTGTGGCTGCAATCATTG

2OAT4g01320 TGATCAAAGGAACATTCCTCTCTGTCATACTAGGCCCACCCATTGTTGCTGCGATAATTT

PPI-AtCPP TGATCAAAGGAACATTCCTCTCTGTCATACTAGGCCCACCCATTGTTGCTGCGATAATTT

BASF TGATCAAAGGAACATTCCTCTCTGTCATACTAGGCCCACCCATTGTTGCTGCGATAATTT

_ TGATCAAAGGAACATTCCTCTCTGTCATACTAGGCCCACCCATTGTTGCTGCGATAATTT
afcl BASF

_ TGATCAAAGGAATACTCCTCTCTGTCATACCTGCCCCTCCTATCGTTGCCGCAATTATTG
PPI-BnCPP

BASF-Corn TGATCAAAGGAATTTTACTATCCATGATATTGGGGCCACCAATCGTGGCTGCTATCATCT
** * ******* * ** ** * *** * ** ** ** ** ** **
** **

30PPI-GmCPP TAATAGTACAG-_____________-_--__--___________________________ BASF-Gm TAATAGTACAG--_____________________--_______________-________ AT4g01320 TCATAGTCCAG-------------------------------------------------PPI-AtCPP TCATAGTCCAG-________________________________________________ 35BASF_AT2 TCATAGTCCAG-------------------------------------------------afcl TCATAGTCCAG-______________--________________________________ BASF_AT1 TCATAGTCCAG-------------------------------------------------PPI-BnCPP TTATAGTTCAG-______-______________-___--_-_--________________ BASF-Corn ACATAGTACAG-------------------------------------------------40 ***** ***

PPI-GmCPP ______________________--____________________________________ BASF-Gm _____________-______________________________________________ AT4g01320 _-___-_-_____---__-______________-_-_-___-__________________ 45PPI-AtCPP -______________________________---_________________--_--____ BASF_AT2 ______________-________________________-___-________________ afcl _______________-_____-______________________________________ BASF_AT1 _________-________________-_______________-___-__-__________ PPI-BnCPP ______________________________-_---__---____--_____-________ 50BASF-Corn ___________________________-________________________________ PPI-GmCPP ------------------------------------AAAGGAGGTCCATACTTGGCCATC

BASF-Gm ------------------------------------AAAGGAGGTCCATACTTGGCCATC

AT4g01320 ------------------------------------AAAGGAGGTCCTTATCTTGCCATC

PPI-AtCPP ------------------------------------AAAGGAGGTCCTTATCTTGCCATC

BASF_AT2 ------------------------------------AAAGGAGGTCCTTATCTTGCCATC

afcl ------------------------------------AAAGGAGGTCCTTATCTTGCCATC

BASF

60_ ------------------------------------AAAGGAGGTCCTTACCTCGCCATC
PPI-BnCPP

BASF-Corn ------------------------------------ATTGGAGGACCTTACCTGGCTATA
* ***** ** ** * ** **

PPI-GmCPP TATCTTTGGGTTTTTACGTTTGGTCTTTCTATTGTGATGATGACCCTTTATCCAGTACTA

BASF-Gm TATCTTTGGGTTTTTACGTTTGGTCTTTCTATTGTGATGATGACCCTTTATCCAGTACTA

65AT4g01320 TATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATACCCGGTCTTG

PPI-AtCPP TATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATACCCGGTCTTG

afcl TATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATACCCGGTCTTG
BASF_AT1 TATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATACCCGGTCTTG
PPZ-BnCPP TATCTGTGGGCATTCATGTTTATCCTGTCTCTAGTGATGATGACTATATACCCTGTTTTG
BASF-Corn TATCTCTGGGGTTTTATGTTTGTATTAGCTCTACTGATGATGACAATATACCCCATTGTG
***** **** ** * **** * ** * ********** * ** **
PPI-GmCPP ATAGCTCCACTCTTCAATAAGTTCACTCCA------------------------------BASF-Gm ATAGCTCCACTCTTCAATAAGTTCACTCCA------------------------------AT4g01320 ATAGCACCGCTCTTCAACAAGTTCACTCCT------------------------------lOAF007269 ATAGCACCGCTCTTCAACAAGTTCACTCCTGTGTGTATTTCTGTCATGGCCATTTTACAA

PPI-AtCPP ATAGCACCGCTCTTCAACAAATTCACTCCT------------------------------BASF_AT2 ATAGCACCGCTCTTCAACAAGTTCACTCCT------------------------------afcl ATAGCACCGCTCTTCAACAAGTTCACTCCT------------------------------BASF ATAGCACCGCTCTTCAACAAGTTCACTCCT------------------------------1S_ ATTGCACCTCTTTTCAACAAGTTCACTCCT------------------------------PPI-BnCPP

BASF-Corn ATAGCTCCTCTGTTCAACAAGTTCACTCCT------------------------------** ** ** ** ***** ** ********

PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ 20AT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ 2SBASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ _ ____________________________________________________________ BASF Corn PPI-GmCPP ----CTTCCAGATGGTCAACTCAGGGAGAAAATCGAGAAACTTGCTTCCTCCCTCAACTA

30BASF-Gm ----CTTCCAGATGGTCAACTCAGGGAGAAAATCGAGAAACTTGCTTCCTCCCTCAACTA

AT4g01320 ----CTTCCAGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTT

PPI-AtCPP ----CTTCCAGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCCCTAAAGTT

BASF ----CTTCCAGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTT

3S_ ----CTTCCAGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTT
afcl BASF_AT1 ----CTTCCAGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTT

PPI-BnCPP ----CTTCCTGATGGAGACCTCCGGGAGAAGATTGAGAAACTTGCTTCTTCTCTAAAGTT

BASF-Corn ----CTTCCTGAAGGAGTCCTCAGGGAAP.AAATAGAGAAGCTGGCAGCTTCCCTCAAGTT
***** ** ** *** **** ** ** ***** ** ** * ** **
**

PPI-GmCPP TCCGTTAAAGAAACTATTTGTTGTCGATGGATCCACAAGATCAAGTCACAGCAATG----BASF-Gm TCCGTTAAAGAAACTATTTGTTGTCGATGGATCCACAAGATCAAGTCACAGCAATG----AT4g01320 TCCTTTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGCAATG----4SPPI-AtCPP TCCTTTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGCAATG----BASF_AT2 TCCTTTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGCAATG----afcl TCCTTTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGCAATG----BASF_AT1 TCCTTTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGCAATG----PPI-BnCPP TCCTCTGAAGAAGCTGTTTGTTGTCGATGGATCTACAAGGTCAAGCCATAGTAATG----SOBASF-Corn TCCTTTGAAAAAGCTTTTCGTGGTAGATGGGTCTACCAGATCAAGCCACAGTAATG----*** * ** ** ** ** ** ** ***** ** ** ** *****
** ** ****

PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ AT1 ____________________________________________________________ BASF

60_ ____________________________________________________________ PPI-BnCPP

BASF-Corn ____________________________________________________________ PPI-GmCPP ----------------CCTATATGTATGGATTCTTCAAGAACAAGAGGATTGTCCCTTAT

BASF-Gm ----------------CCTATATGTATGGATTCTTCAAGAACAAGAGGATTGTCCTTTAT

6SAT4g01320 ----------------CTTACATGTATGGTTTCTTTAAGAACAAAAGGATTGTTCTTTAT

PPI-AtCPP ----------------CTTACATGTATGGTTTCTTTAAGAACAAAAGGATTGTTCTTTAT

afcl ----------------CTTACATGTATGGTTTCTTTAAGAACAAAAGGATTGTTCTTTAT

BASF ----------------CTTACATGTATGGTTTCTTTAAGAACAAAAGGATTGTTCTTTAT

_ ----------------CTTACATGTATGGTTTCTTCAAGAACAAAAGGATTGTTCTTTAT
PPI-BnCPP

BASF-Corn ----------------CCTACATGTATGGTTTTTTCAAGAACAAGCGCATAGTACTCTAT

* ** ******** ** ** ******** * ** ** * ***

PPI-GmCPP GACACATTAATTCAACAG------------------------------------------BASF-Gm GACACATTAATTCAACAG------------------------------------------AT4g01320 GATACGTTGATTCAGCAG------------------------------------------1O PPI-AtCPP GATACGTTGATTCAGCAG------------------------------------------BASF GATACGTTGATTCAGCAG------------------------------------------_ GATACGTTGATTCAGCAG------------------------------------------afcl BASF_AT1 GATACGTTGATTCAGCAG------------------------------------------PPI-BnCPP GACACATTGATTCAGCAG------------------------------------------1S BASF-Corn GACACATTGATTCAGCAG------------------------------------------** ** ** ***** ***

PPI-GmCPP ______________-_____________________________TGCAAAGACGATGAGG

BASF-Gm --------------------------------------------TGCAAAGACGATGAGG

20 AT4g01320 --------------------------------------------TGCAAGAATGAGGATG

PPI-AtCPP ____________________________________________TGCAAGAATGAGGATG

BASF --------------------------------------------TGCAAGAATGAGGATG

_ --------------------------------------------TGCAAGAATGAGGATG
afcl 2S BASF_AT1 --------------------------------------------TGCAAGAATGAGGATG

PPI-BnCPP __________________-_______________-_________TGCCAGAATGAGAATG

BASF-Corn --------------------------------------------TGTAGCAATGAGGATG
** * **

30 PPI-GmCPP AAATTGTTGCTGTTATTGCCCATGAGTTGGGACACTGGAAGCTCAACCATACTGTGTACA

BASF-Gm AAATTGTTGCTGTTATTGCCCATGAGTTGGGACACTGGAAGCTCAACCATACTGTGTACA

AT4g01320 AAATTGTGGCGGTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACT

PPI-AtCPP AAATTGTGGCGGTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACT

3S BASF_AT2 AAATTGTGGCGGTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACT

afcl AAATTGTGGCGGTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACT

BASF_AT1 AAATTGTGGCGGTTATTGCACACGAGCTTGGACATTGGAAACTGAATCACACTACATACT

PPI-BnCPP AAATTGTGGCGGTTATTGCACACGAGCTGGGACACTGGAAGCTGAATCACACTACATACT

BASF-Corn AGATAGTTTCTGTTATAGCACATGAACTTGGACACTGGAAACTCAATCATACTGTCTATT

40 * ** ** * ***** ** ** ** * ***** ***** ** **
** *** **

PPI-GmCPP CATTTGTTGCTATGCAG-------------------------------------------BASF-Gm CATTTGTTGCTATGCAG-------------------------------------------AT4g01320 CGTTCATTGCAGTTCAA-------------------------------------------4S PPI-AtCPP CGTTCATTGCAGTTCAA-------------------------------------------BASF_AT2 CGTTCATTGCAGTTCAA-------------------------------------------afcl CGTTCATTGCAGTTCAA-------------------------------------------BASF_AT1 CGTTCATTGCAGTTCAA-------------------------------------------PPI-BnCPP CGTTCATTGCTGTTCAA-------------------------------------------SO BASF-Corn CCTTTGTAGCTGTCCAG-------------------------------------------* ** * ** * **

PPI-GmCPP __________-______-_________________________-_______ATTCTTACA

BASF-Gm _-_-_____-_________-___-_____-_____________________ATTCTTACA

AT4g01320 __________________________________-________________ATCCTTGCC

PPI-AtCPP ___________-______-____-_________________-_________p~TCCTTGCC

BASF ______________________-____________________________ATCCTTGCC

_ ____-______________________________________________ATCCTTGCC
afcl BASF ___________________-__________-_____________-______p~TCCTTGCC

60 _ --__-________-_--__-_-_____________________________ATCCTTGCC
PPI-BnCPP

BASF-Corn _________________________________-_________-_______CTGCTTATG

* ***

PPI-GmCPP CTTCTACAATTTGGAGGATATACACTAGTGCGAAATTCAGCTGATCTGTATCGAAGCTTT

6S BASF-Gm CTTCTACAATTTGGAGGATATACACTAGTGCGAAATTCAGCTGATCTGTATCGAAGCTTT

AT4g01320 TTCTTACAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTC

PPI-AtCPP TTCTTACAATTTGGAGGATACACTCTTCTCAGAAACTCCACTGATCTCTTCAGGAGTTTC

BASF_AT2 TTCTTACAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTC
afcl TTCTTACAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTC
BASF_AT1 TTCTTACAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTC
PPI-BnCPP TTCTTGCAATTTGGAGGATACACTCTTGTCAGAAACTCCACTGATCTCTTCAGGAGTTTT
BASF-Corn TTTCTTCAATTTGGAGGATATACTCTAGTAAGGAGCTCCAAAGATCTATTTGGAAGTTTT
* * ************** ** ** * * * ** ***** * * ** **
PPI-GmCPP GGGTTTGATACGCAGCCAGTCCTCATTGGGCTCATCATATTTCAG---------------BASF-Gm GGGTTTGATACGCAGCCAGTCCTCATTGGGCTCATCATATTTCAG---------------lO AT4g01320 GGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG---------------PPI-AtCPP GGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG---------------BASF_AT2 GGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG---------------afcl GGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG---------------IS BASF_AT1 GGATTTGATACACAGCCTGTTCTCATTGGTTTGATCATATTTCAG--------------PPI-BnCPP GGTTTTGATACACAACCAGTTCTCATTGGTTTGATCATATTTCAG--------------BASF-Corn GGCTTCAAGGACCAGCCAGTAATAATTGGATTGATCATTTTCCCG--------------** ** * ** ** ** * ***** * ***** **
20 PPI-GmCPP
BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ 25BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ BASF-Corn ____________________________________________________________ 30PPI-GmCPP ---------------------------CATACTGTAATCCCACTTCAGCAATTGGTCAGC

BASF-Gm ---------------------------CATACTGTAATCCCACTTCAGCAATTGGTCAGC

AT4g01320 ---------------------------CACACTGTAATACCACTGCAACATCTAGTAAGC

PPI-AtCPP ---------------------------CACACTGTAATACCACTGCAACATCTAGTAAGC

_ ---------------------------CACACTGTAATACCACTGCAACATCTAGTAAGC
afcl BASF_AT1 ---------------------------CACACTGTAATACCACTGCAACATCCAGTAAGC

PPI-BnCPP ---------------------------CACACTGTAATACCACTTCAACACCTAGTAAGC

BASF-Corn ---------------------------CACACCATAATACCCATCCAACACCTTCTGAGC

40 ** ** **** ** * ** ** * ***

PPI-GmCPP TTTGGTCTGAACCTAGTCAGCCGATCATTTGAATTTCAGG--------------------BASF-Gm TTTGGTCTGAACCTAGTCAGCCGATCATTTGAATTTCAGG--------------------AT9g01320 TTTGGCCTGAACCTCGTTAGTCGAGCGTTTGAGTTTCAGG--------------------PPI-AtCPP TTTGGCCTGAACCTCGTTAGTCGAGCGTTTGAGTTTCAGG--------------------BASF_AT2 TTTGGCCTGAACCTCGTTAGTCGAGCGTTTGAGTTTCAGG--------------------afcl TTTGGCCTGAACCTCGTTAGTCGAGCGTTTGAGTTTCAGG--------------------BASF

50_ TTTGACCTCAACCTTGTTAGTCGAGCGTTTGAGTTTCAGG--------------------PPI-BnCPP

BASF-Corn TTTCGCCTGAACCTTGTCAGCAGAGCATTTGAATTTCAGG--------------------*** ** ***** ** ** ** * ***** *******

PPI-GmCPP ____________________________________________________________ BASF-Gm ______________________________________________________-_____ 5SAT4g01320 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ 6OBASF_AT1 ______________________________________________________-_____ PPI-BnCPP ____________________________________________________________ BASF-Corn ____________________________________________________________ PPI-GmCPP ------------CTGATGGCTTTGCCAAGAAGCTTGGATATGCATCTGGATTACGCGGTG

65BASF-Gm ------------CTGATGGCTTTGCCAAGAAGCTTGGATATGCATCTGGATTACGCGGTG

AT4g01320 ------------CTGATGCTTTTGCTGTGAAGCTTGGCTATGCAAAAGATCTTCGTCCTG

PPI-AtCPP ------------CTGATGCTTTTGCTGTGAAGCTTGACTATGCAAAAGATCTTCGTCCTG

BASF_AT2 ------------CTGATGCTTTTGCTGTGAAGCTTGGCTATGCAAAAGATCTTCGTCCTG
afcl ------------CTGATGCTTTTGCCGTGAAGCTTGGCTATGCAAAAGATCTTCGTCCTG
BASF_AT1 ------------CTGATGCTTTTGCTGTGAAGCTTGGCTATGCAAAAGATCTTCGTCCTA
PPI-BnCPP ------------CTGATGCTTTTGCAGTGAATCTTGGTTATGCAAAGGATCTACGTCCTG
BASF-Corn ------------CTGATGCCTTTGCCAAGAACCTTGGATATGCCCCTCAGCTCCGAGCAG
****** ***** *** **** ***** * **
PPI-GmCPP GTCTTGTGAAACTACAGG------------------------------------------BASF-Gm GTCTTGTGAAACTACAGG------------------------------------------lOAT4g01320 CTCTAGTGAAACTACAGGTCAGAGAAGATAACAACAGAACACAAACTGTTACCTCAATTT

PPI-AtCPP CTCTAGTGAAACTACAGG------------------------------------------BASF_AT2 CTCTAGTGAAACTACAGG------------------------------------------afcl CTCTAGTGAAACTACAGG------------------------------------------15BASF_AT1 CTCTAGTGAAACTACAGG------------------------------------------PPI-BnCPP CCCTAGTGAAGCTACAGG------------------------------------------BASF-Corn CCCTTGTTAAACTACAGG------------------------------------------** ** ** *******

PPI-GmCPP ------------------------------------------AGGAGAATCTGTCAGCTA

20BASF-Gm ------------------------------------------AGGAGAATCTGTCAGCTA

AT4g01320 GTGTCACACACTTAAATGGATTTTTTGTTGGGATTTTGCAGGAAGAGAACTTATCAGCAA

PPI-AtCPP __________________________________________~GAGAACTTATCAACAA

BASF ------------------------------------------AAGAGAACTTATCAGCAA

2S_ ------------------------------------------AAGAGAACTTATCAGCAA
afcl BASF_AT1 ------- . --------------------------------AAGAGAACTTATCAGCAA

PPI-BnCPP ------------------------------------------AAGAGAACTTATCAGCGA

BASF-Corn ------------------------------------------AGGAGAACTTGTCTGCGA
* ***** * **

PPI-GmCPP TGAATACAGATCCTTGGTACTCTGCTTATCACTATTCTCATCCTCCCCTTGTTGAAAGAT

BASF-Gm TGAATACAGATCCTTGCT--CGTGCCG---------------------------------AT4g01320 TGAACACTGATCCATTGTACTCAGCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGC

3SPPI-AtCPP TGAACACTGATCCATTGTACTCAGCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGC

BASF_AT2 TGAAAACTGATCTATTGTACTCAGCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGC

afcl TGAACACTGATCCATTGCACTCAGCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGC

BASF_AT1 TGAATACTGATCCATTGTACTCAGCTTATCACTACTCACATCCTCCTCTTGTTGAAAGGC

PPI-BnCPP TGAACACAGACCCATTGTACTCAGCTTATCACTACTCACACCCTCCTCTTGTAGAGAGGC

40BASF-Corn TGAACACCGATCCTTGGTATTCGGCATATCACTACTCCCACCCACCACTCGTCGAGAGGC
**** ** ** * * **

PPI-GmCPP TGGCCGCGCTGGACGA---ACCGGATAAGAAGGAAGACTAA-------------------BASF-Gm ____________________________________________________________ 45AT4g01320 TTCGAGCCATTGATGG---AGAAGACAAGAAGACAGATTAA-------------------PPI-AtCPP TTCGAGCCACTGATGG---AGAAGACAAGAAGACAGATTAA-------------------BASF_AT2 TTCGAGCCATTGATGG---AGAAGACAAGAAGACAGATTAA-------------------afcl TTCGAGCCATTGATGG---AGAAGACAAGAAGACAGATTAA-------------------50BASF_AT1 TTCGAGCCATTGATGG---AGAAGACAAGAAGACAGATTAA-------------------PPI-BnCPP TTCGAGCCATTGATGG---AGAAGACAAGAAGACAGATTAA-------------------BASF-Corn TGCAAGCTTTGGAAGATTCAGACGACAAAAAAGAAGATTAGTCGATCCTTGTATGAGGTT

55 PPI-GmCPP
BASF-Gm ___________________________________-______________________-_ AT4g01320 ____________________________________________________________ AF007269 _____________-______________________________________________ PPI-AtCPP ____________________________________________________________ 6O BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 _______________________-_____________________________-__-___ PPI-BnCPP ______-_____________________________________________________ BASF-Corn TACATATGGATTTTTCCCTGCCACATGCACACCGATTCAGTGCTTGGATGGTGAGGGTTT
PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ AF007269 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl _______________________________________-____________________ S BASF_AT1 ____________________________________________________________ PPI-BnCPP ____________________________________________________________ BASF-Corn TGACATAGGAGTGTTGTCAAAGCTTTAGAGTGCATCTTTCGGTCAGGTGCAACAGCCTTT
PPI-GmCPP
BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ AF007269 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl __________________________________________________________-_ BASF_AT1 _______________________________-____________________________ PPI-BnCPP ____________________________________-_______________________ BASF-Corn CGGTCATTGAGACATATAAGCGAATTAGCTATTAAAAAAAACAGAACTGTTGCATCAAAA
PPI-GmCPP ____________________________________________________________ BASF-Gm ____________________________________________________________ AT4g01320 ____________________________________________________________ AF007269 ____________________________________________________________ PPI-AtCPP ____________________________________________________________ BASF_AT2 ____________________________________________________________ afcl ____________________________________________________________ BASF_AT1 ____________________________________________________________ 3o PPI-BnCPP ____________________________________________________________ BASE-Corn AA GAAAC G
PPI-GmCPP ____________________________________________________ BASF-Gm ____________________________________________________ AT4g01320 ____________________________________________________ AF007269 ____________________________________________________ PPI-AtCPP ____________________________________________________ BASF_AT2 ____________________________________________________ afcl ____________________________________________________ BASF_AT1 ____________________________________________________ PPI-BnCPP ____________________________________________________ BASF-Corn AAAAAGTGCTCTGCGTTGTTACCACTGCTTGCCCTATAGTGATCGTATCAGA
Table 6B. ClustalW Amino Acid Analysis of CaaX Prenyl Protease 1: PPI-AtCPP SEQ ID N0:2 2: PPI-BnCPP SEQ ID NO:15 3: PPI-GmCPPSEQ ID N0:18 4: BASF AT1 SEQ ID N0:22 5: BASF AT2 SEQ ID N0:24 6: BASF-Corn SEQ ID N0:26 7: BASF-Gm SEQ ID N0:28 8: AFCI SEQ
ID N0:30 9: AT4g01320 SEQ ID N0:32 10: AF007269 SEQ ID N0:34 PPI-GmCPP MAFPYMEAWGFMILMYIFETYLDVRQHRALKLPTLPKTLEG-------VISQEKFEKSR

BASF-Gm MAFPYMEAVVGFMILMYIFETYLDVRQHRALKLPTLPKTLEG-------VISQEKFEKSR

AT4g-AtCPP MAIPFMETVVGFMIVMYIFETYLDLRQLTALKLPTLPKTLVGVISQEKFEKSRAYRDIIT

BASF MAIPFMETVVGFMIVMYIFETYLDLRQLTALKLPTLPKTLVG-------VISQEKFEKSR

_ MAIPFMETWGFMIVMYIFETYLDLRQLTALKLPTLPKTLVG-------VISQEKFEKSR

BASF_AT1 MAIPFMETVVGFMIVMYIFETYLDLRQLTALKLPTLPKTLVG-------VISQEKFEKSR

PPI-AtCPP MAIPFMETVVGFMIVMYIFETYLDLRQLTALKLPTLPKTLVG-------VISQEKFEKSR

PPI-BnCPP MAIPFMETWGFMIVMYVFETYLDLRQHTALKLPTLPKTLVG-------VISQEKFEKSR

BASF-Corn ______________________________________________________-_____ PPI-GmCPP AYSLDKSHFHFVHEFVTIVTDSTILYFGVLPWFWKKSGDFMTIAGFNAENEILHTLAFLA

BASF-Gm AYSLDKSHFHFVHEFVTIVTDSTILYFGVLPWFWKKSGDFMTIAGFNAENEILHTLAFLA

AF007269 ____________________________________________________________ AT4g-AtCPP ENFNICSYFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLA

BASF AYSLDKSYFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLA

_ AYSLDKSYFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLA

IS BASF_ATl AYSLDKSYFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLA

PPI-AtCPP AYSLDKSYFHFVHEFVTILMDSAILFFGILPWFWKMSGAVLPRLGLDPENEILHTLSFLA

PPI-BnCPP AYSLDKSHFHFVHEFVTILMDSAILFFGILPWFWKISGGFLPMVGLDPENEILHTLSFLA

BASF-Corn -------------------------------------------TRLSAENEIIHTLAFLA

PPI-GmCPP GLMIWSQITDLPFSLYSTFVIEARHGFNKQTPWLFFRDMLKGIFLSVIIGPPIVAAIIVI

BASF-Gm GLMIWSQITDLPFSLYSTFVIEARHGFNKQTPWLFFRDMLKGIFLSVIIGPPIVAAIIVI

AF007269 --------TDLPFSLYSTFVIESRHGE'NKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFI

AT4g-AtCPP GVMTWSQITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFI

BASF_AT2 GVMTWSQITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFI

BASF GVMTWSQITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFI

_ GVMTWSQITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGTFLSVILGPPIVAAIIFI
PPI-AtCPP

PPI-BnCPP GLMTWSQITDLPFSLYSTFVIESRHGFNKQTIWMFIRDMIKGILLSVIPAPPIVAAIIVI

BASF-Corn GSMVWSQITDLPFSLYSTFVIEARHGFNKQTIWLFIRDMIKGILLSMILGPPIVAAIIYI

**************.******** *.*.***.** .**.* ********

PPI-GmCPP VQKGGPYLAIYLWVFTFGLSIVMMTLYPVLIAPLFNKFTPLPDGQLREKIEKLASSLNYP

BASF-Gm VQKGGPYLAIYLWVFTFGLSIVMMTLYPVLIAPLFNKFTPLPDGQLREKIEKLASSLNYP

AT4g-AtCPP VQKGGPYLAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFP

BASF_AT2 VQKGGPYLAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFP

BASF VQKGGPYLAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFP

_ VQKGGPYLAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFP
4O PPI-AtCPP

PPI-BnCPP VQKGGPYLAIYLWAFMFILSLVMMTIYPVLIAPLFNKFTPLPDGDLREKIEKLASSLKFP

BASF-Corn VQIGGPYLAIYLWGFMFVLALLMMTIYPIVIAPLFNKFTPLPEGVLREKIEKLAASLKFP

** ********** * * *...***.**..************.*
*********.**..*

PPI-GmCPP LKKLFWDGSTRSSHSNAYMYGFFKNKRIVPYDTLIQQCKDDEEIVAVIAHELGHWKLNH

BASF-Gm LKKLFVVDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKDDEEIVAVIAHELGHWKLNH

SO AT4g-AtCPPLKKLFVVDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKNEDEIVAVIAHELGHWKLNH

BASF_AT2 LKKLE'WDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKNEDEIVAVIAHELGHWKLNH

BASF_AT1 LKKLF'WDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKNEDEIVAVIAHELGHWKLNH

PPI-AtCPP LKKLFVVDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCKNEDEIVAVIAHELGHWKLNH

SS PPI-BnCPP LKKLFVVDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCQNENEIVAVIAHELGHWKLNH

BASF-Corn LKKLFWDGSTRSSHSNAYMYGFFKNKRIVLYDTLIQQCSNEDEIVSVIAHELGHWKLNH

****************************** ******** . .***.*************

PPI-GmCPP TVYTFVAMQILTLLQFGGYTLVRNSADLYRSFGFDTQPVLIGLIIFQHTVIPLQQLVSFG

60 BASF-Gm TVYTFVAMQILTLLQFGGYTLVRNSADLYRSFGFDTQPVLIGLIIFQHTVIPLQQLVSFG

AT4g-AtCPP TTYSFIAVQILAFLQFGGYTLVRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFG

BASF_AT2 TTYSFIAVQILAFLQFGGYTLVRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFG

65 BASF_AT1 TTYSFIAVQILAFLQFGGYTLVRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHPVSFG

PPI-AtCPP TTYSFIAVQILAFLQFGGYTLLRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFG

PPI-BnCPP TTYSFIAVQILAFLQFGGYTLVRNSTDLFRSFGFDTQPVLIGLIIFQHTVIPLQHLVSFD

BASF-Corn TVYSFVAVQLLMFLQFGGYTLVRSSKDLFGSFGFKDQPVIIGLIIFPHTIIPIQHLLSFR

* *.*.*. **.**.*. .**

PPI-GmCPP LNLVSRSFEFQADGFAKKLGYASGLRG---------------------------------BASF-Gm LNLVSRSFEFQADGFAKKLGYASGLRG---------------------------------AT4g-AtCPP LNLVSRAFEFQADAFAVKLGYAKDLR-------PALV----KLQVREDNNRTQTVTSICV

BASF_AT2 LNLVSRAFEFQADAFAVKLGYAKDLR-------PALV----KLQE---------------BASF LNLVSRAFEFQADAFAVKLGYAKDLRPTLVKLQ---------------------------IO_ LNLVSRAFEFQADAFAVKLDYAKDLRPALVKLQ---------------------------PPI-AtCPP

PPI-BnCPP LNLVSRAFEFQADAFAVNLGYAKDLRP---------------------------------BASF-Corn LNLVSRAFEFQADAFAKNLGYAPQLR----------------------------------******.****** ** .* ** **

15PPI-GmCPP ------GLVKLQEENLSAMNTDPWYSAYHYSHPPLVERLAALDEPDKKED-BASF-Gm ------GLVKLQEENLSAMNTDPCSC-------------------------AT4g-AtCPP THLNGFFVGILQEENLSAMNTDPLYSAYHYSHPPLVERLRAIDGEDKKTD-BASF -------------ENLSAMNTDPLYSAYHYSHPPLVERLRAIDGEDKKTD-2O_ -------------ENLSAMNTDPLHSAYHYSHPPLVERLRAIDGEDKKTD-BASF_AT1 ------------EENLSAMNTDPLYSAYHYSHPPLVERLRAIDGEDKKTD-PPI-AtCPP ------------EENLSTMNTDPLYSAYHYSHPPLVERLRATDGEDKKTD-PPI-BnCPP ------ALVKLQEENLSAMNTDPLYSAYHYSHPPLVERLRAIDGEDKKTD-BASF-Corn -----AALVKLQEENLSAMNTDPWYSAYHYSHPPLVERLQALEDSDDKKED

25 ****;***** .

Example 4: Plant Transformation 3o Arabidopsis transgenic plants were made by the method of dipping flowering plants into an Agrobacterium culture, based on the method of Andrew Bent in, Clough SJ and Bent AF, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Wild type plants were grown under standard conditions until the plant has both developing flowers and open flowers. The plant was 35 inverted for 2 minutes into a solution of Agrobacterium culture carrying the appropriate gene construct. Plants were then left horizontal in a tray and kept covered for two days to maintain humidity and then righted and bagged to continue growth and seed development. Mature seed was bulk harvested.
Transformed T1 plants were selected by germination and growth on MS plates 40 containing 50 ~g/ml kanamycin. Green, kanamycin resistant (KanR) seedlings were identified after 2 weeks growth and transplanted to soil. Plants were bagged to ensure self fertilization and the T2 seed of each plant harvested separately. During growth of T1 plants leaf samples were harvested, DNA extracted and Southern blot and PCR
analysis performed.

T2 seeds were analysed for KanR segregation. From those lines that showed a 3:1 resistant phenotype, surviving T2 plants were grown, bagged during seed set, and T3 seed harvested from each line. T3 seed was again used for KanR segregation analysis and those lines showing 100% KanR phenotype were selected as homozygous lines.
Further molecular and physiological analysis was done using T3 seedlings.
Transgenic Brassica napus, Glycine max and Zea maize plants were produced using Agrobacterium mediated transformation of cotyledon petiole tissue. Seeds were sterilized as follows. Seeds were wetted with 95% ethanol for a short period of time such as 15 seconds. Approximately 30 ml of sterilizing solution I was added (70%
Javex , to 100p,1 Tween20) and left for approximately 15 minutes. Solution I was removed and replaced with 30 ml of solution II (0.25% mecuric chloride, 100p1 Tween20) and incubated for about 10 minutes. Seeds were rinsed with at least 500 ml double distilled sterile water and stored in a sterile dish. Seeds were germinated on plates of medium, pH 5.8, supplemented with 1 % sucrose and 0.7% agar. Fully expanded 1 s cotyledons were harvested and placed on Medium I (Murashige minimal organics (MMO), 3% sucrose, 4.5 mg/L benzyl adenine (BA), 0.7% phytoagar, pH5.8). An Agrobacterium culture containing the nucleic acid construct of interest was grown for 2 days in AB Minimal media. The cotyledon explants were dipped such that only the cut portion of the petiole is contacted by the Agrobacterium solution. The explants were then 2o embedded in Medium I and maintained for 5 days at 24°C, with 16,8 hr light dark cycles.
Explants were transferred to Medium II (Medium I, 300 mg/L timentin,) for a further 7 days and then to Medium III (Medium II, 20 mg/L kanamycin). Any root or shoot tissue which had developed at this time was dissected away. Transfer explants to fresh plates of Medium III after 14 -21 days. When regenerated shoot tissue developed 25 the regenerated tissue was transferred to Medium IV (MMO, 3% sucrose, 1.0%
phytoagar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin). Once healthy shoot tissue developed shoot tissue dissected from any callus tissue was dipped in l OX IBA
and transferred to Medium V (Murashige and Skooge (MS), 3% sucrose, 0.2 mg/L
indole butyric acid (IBA), 0.7% agar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin) for 3o rooting. Healthy plantlets were transferred to soil. The above method, with or without modifications, is suitable for the transformation of numerous plant species including Glycine max, Zea maize and cotton.

Transgenic Glycine max, Zea maize and cotton can be produced using Agrobacterium-based methods which are known to one of skill in the art.
Alternatively one can use a particle or non-particle biolistic bombardment transformation method. An example of non-particle biolistic transformation is given in U.S. Patent Application 20010026941. This method has been used to produce transgenic Glycine max and Zea maize plants. Viable plants are propagated and homozygous lines are generated.
Plants are tested for the presence of drought tolerance, physiological and biochemical phenotypes as described elsewhere.
The following table identifies the constructs and the species which they have l0 been transformed.
Table 7 Transformation List SEQ ID NO: Construct Species Transformed 4 pBII121-AtCPP A. thaliana, B. napes 5 pBII121-HP-AtCPP A. thaliana 36 pRD29A-AtCPP A. thaliana, B. napes 37 pRD29A-HP-AtCPP A. thaliana 39 MuA-AtCPP Glycine max, Zea mays Non-limiting examples of vector constructs suitable for plant transformation are given in 2o SEQ ID NO: 4, 5, 35-53.
SEQ ID N0:4 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaggatccatggcgattcctttcatggaaaccgtcgtg ggttttatgatagtgatgtacatttttgagacgtatttggatctgaggcaactcactgc tctcaagcttccaactctcccgaaaaccttggttggtgtaattagccaagagaagtttg agaaatcacgagcatacagtcttgacaaaagctattttcactttgttcatgagtttgta actatacttatggactctgcaattttgttctttgggatcttgccttggttttggaagat gtctggagctgttttaccgaggttgggccttgatccggagaatgaaatactgcatactc tttcattcttggctggtgttatgacatggtcacagatcactgatttgccattttctttg tactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatgtt cattagggacatgatcaaaggaacattcctctctgtcatactaggcccacccattgttg ctgcgataattttcatagtccagaaaggaggtccttatcttgccatctatctgtgggca ttcatgtttatcctgtctctagtgatgatgactatatacccggtcttgatagcaccgct cttcaacaaattcactcctcttccagatggagacctccgggagaagattgagaaacttg cttcttccctaaagtttcctttgaagaagctgtttgttgtcgatggatctacaaggtca agccatagcaatgcttacatgtatggtttctttaagaacaaaaggattgttctttatga tacgttgattcagcagtgcaagaatgaggatgaaattgtggcggttattgcacacgagc ttggacattggaaactgaatcacactacatactcgttcattgcagttcaaatccttgcc ttcttacaatttggaggatacactcttctcagaaactccactgatctcttcaggagttt cggatttgatacacagcctgttctcattggtttgatcatatttcagcacactgtaatac cactgcaacatctagtaagctttggcctgaacctcgttagtcgagcgtttgagtttcag gctgatgcttttgctgtgaagcttgactatgcaaaagatcttcgtcctgctctagtgaa actacaggaagagaacttatcaacaatgaacactgatccattgtactcagcttatcact actcacatcctcctcttgttgaaaggcttcgagccactgatggagaagacaagaagaca gattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagat tgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaag catgtaa~taattaacatgtaatgcatgacgttatttatgagatgggtttttatgattag agtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactagg ataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcac atccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgcca cgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattt agtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgat ttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgggg caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatca gctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgc aatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:4 is the nucleic acid sequence of pBI121-AtCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter and bolded sequence is the AtCPP sense sequence.
2o SEQ ID NO:S
gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaggatcctcccaatgtccaagctcgtgtgcaataacc gccacaatttcatcctcattcttgcactgctgaatcaacgtatcataaagaacaatcct tttgttcttaaagaaaccatacatgtaagcattgctatggcttgaccttgtagatccat cgacaacaaacagcttcttcaaaggaaactttagggaagaagcaagtttctcaatcttc tcccggaggtctccatctggaagaggagtgaatttgttgaagagcggtgctatcaagac cgggtatatagtcatcatcactagagacaggataaacatgaatgcccacagatagatgg caagataaggacctcctttctggactatgaaaattatcgcagcaacaatgggtgggcct agtatgacagagaggaatgttcctttgatcatgtccctaatgaacatccatattgtttg tttgttgaacccatgccgagactcgatcacgaaagttgagtacaaagaaaatggcaaat cagtgatctgtgaccatgtcataacaccagccaagaatgaaagagtatgcagtatttca ttctccggatcaaggcccaacctcggtaaaagaggatccccATCTACCCGCTTCGCGTC
GGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTA
CTTTACTGGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACG
TGCTGATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACC
TCGCATTACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGT
GATTGATGAAACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGG

GCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCG
CACTTACAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGAT
GTGGAGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGC
CACTGGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTA
ATGTTCTGCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAA
CCGTTATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGG
AAAAAGAACTTCTGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTAT
CAGTGTGCATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGG
TGAACAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCG
GTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTG
CAAAAACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATG
AATCAACAACTCTCCTGGCGCACCATCGTCGGCTACAGCCTCGGGAATTGCTACCGAGC
TCttttaccgaggttgggccttgatccggagaatgaaatactgcatactctttcattct tggctggtgttatgacatggtcacagatcactgatttgccattttctttgtactcaact ttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatgttcattaggga catgatcaaaggaacattcctctctgtcatactaggcccacccattgttgctgcgataa ttttcatagtccagaaaggaggtccttatcttgccatctatctgtgggcattcatgttt atcctgtctctagtgatgatgactatatacccggtcttgatagcaccgctcttcaacaa attcactcctcttccagatggagacctccgggagaagattgagaaacttgcttcttccc taaagtttcctttgaagaagctgtttgttgtcgatggatctacaaggtcaagccatagc aatgcttacatgtatggtttctttaagaacaaaaggattgttctttatgatacgttgat tcagcagtgcaagaatgaggatgaaattgtggcggttattgcacacgagcttggacatt ~qgagctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaa tcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatg taataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtc ccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataa attatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgtttta caacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcc ccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagt tgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgccacgtt cgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtg ctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggcca tcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtgg actcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttat aagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctggggcaaa ccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctg ttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatg tgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID NO:S is the nucleic acid sequence of pBI121-HP-AtCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter and bolded sequence is the AtCPP anti-sense sequence. Sequence in upper case is the truncated GUS fragment. Sequence in bold and underlined is the AtCPP sense sequence.
SEQ ID N0:35 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct l0 cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca ;~cgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaggatccTTAATCTGTCTTCTTGTCTTCTCCATCAGT
GGCTCGAAGCCTTTCAACAAGAGGAGGATGTGAGTAGTGATAAGCTGAGTACAATGGAT
CAGTGTTCATTGTTGATAAGTTCTCTTCCTGTAGTTTCACTAGAGCAGGACGAAGATCT
TTTGCATAGTCAAGCTTCACAGCAAAAGCATCAGCCTGAAACTCAAACGCTCGACTAAC
GAGGTTCAGGCCAAAGCTTACTAGATGTTGCAGTGGTATTACAGTGTGCTGAAATATGA
TCAAACCAATGAGAACAGGCTGTGTATCAAATCCGAAACTCCTGAAGAGATCAGTGGAG
TTTCTGAGAAGAGTGTATCCTCCAAATTGTAAGAAGGCAAGGATTTGAACTGCAATGAA
CGAGTATGTAGTGTGATTCAGTTTCCAATGTCCAAGCTCGTGTGCAATAACCGCCACAA
TTTCATCCTCATTCTTGCACTGCTGAATCAACGTATCATAAAGAACAATCCTTTTGTTC
TTAAAGAAACCATACATGTAAGCATTGCTATGGCTTGACCTTGTAGATCCATCGACAAC
AAACAGCTTCTTCAAAGGAAACTTTAGGGAAGAAGCAAGTTTCTCAATCTTCTCCCGGA
GGTCTCCATCTGGAAGAGGAGTGAATTTGTTGAAGAGCGGTGCTATCAAGACCGGGTAT
ATAGTCATCATCACTAGAGACAGGATAAACATGAATGCCCACAGATAGATGGCAAGATA
AGGACCTCCTTTCTGGACTATGAAAATTATCGCAGCAACAATGGGTGGGCCTAGTATGA
CAGAGAGGAATGTTCCTTTGATCATGTCCCTAATGAACATCCATATTGTTTGTTTGTTG

AACCCATGCCGAGACTCGATCACGAAAGTTGAGTACAAAGAAAATGGCAAATCAGTGAT
CTGTGACCATGTCATAACACCAGCCAAGAATGAAAGAGTATGCAGTATTTCATTCTCCG
GATCAAGGCCCAACCTCGGTAAAACAGCTCCAGACATCTTCCAAAACCAAGGCAAGATC
CCAAAGAACAAAATTGCAGAGTCCATAAGTATAGTTACAAACTCATGAACAAAGTGAAA
ATAGCTTTTGTCAAGACTGTATGCTCGTGATTTCTCAAACTTCTCTTGGCTAATTACAC
CAACCAAGGTTTTCGGGAGAGTTGGAAGCTTGAGAGCAGTGAGTTGCCTCAGATCCAAA
TACGTCTCAAAAATGTACATCACTATCATAAAACCCACGACGGTTTCCATGAAAGGAAT
CGCCATcccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagat tgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaag catgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattag agtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactagg ataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcac atccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgcca cgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattt agtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgat ttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgggg caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatca gctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgc aatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:35 is the nucleic acid sequence of pBI121-antisense-AtCPP.
Italicized sequences are the right and left border repeats. Underlined sequence is the 35S
promoter. Sequence in upper case is the AtCPP anti-sense sequence.
SEQ ID N0:36 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat CgCCgCtCCCgattCgCagCgCatCgCCttCtatCgCCttCttgaCgagttCttCtgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatccatggcgattcctttcatggaaacc gtcgtgggttttatgatagtgatgtacatttttgagacgtatttggatctgaggcaact cactgctctcaagcttccaactctcccgaaaaccttggttggtgtaattagccaagaga agtttgagaaatcacgagcatacagtcttgacaaaagctattttcactttgttcatgag tttgtaactatacttatggactctgcaattttgttctttgggatcttgccttggttttg gaagatgtctggagctgttttaccgaggttgggccttgatccggagaatgaaatactgc atactctttcattcttggctggtgttatgacatggtcacagatcactgatttgccattt tctttgtactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatg gatgttcattagggacatgatcaaaggaacattcctctctgtcatactaggcccaccca ttgttgctgcgataattttcatagtccagaaaggaggtccttatcttgccatctatctg tgggcattcatgtttatcctgtctctagtgatgatgactatatacccggtcttgatagc accgctcttcaacaaattcactcctcttccagatggagacctccgggagaagattgaga aacttgcttcttccctaaagtttcctttgaagaagctgtttgttgtcgatggatctaca aggtcaagccatagcaatgcttacatgtatggtttctttaagaacaaaaggattgttct ttatgatacgttgattcagcagtgcaagaatgaggatgaaattgtggcggttattgcac acgagcttggacattggaaactgaatcacactacatactcgttcattgcagttcaaatc cttgccttcttacaatttggaggatacactcttctcagaaactccactgatctcttcag gagtttcggatttgatacacagcctgttctcattggtttgatcatatttcagcacactg taataccactgcaacatctagtaagctttggcctgaacctcgttagtcgagcgtttgag tttcaggctgatgcttttgctgtgaagcttgactatgcaaaagatcttcgtcctgctct agtgaaactacaggaagagaacttatcaacaatgaacactgatccattgtactcagctt atcactactcacatcctcctcttgttgaaaggcttcgagccactgatggagaagacaag aagacagattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttct taagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattac gttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttat gattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct tcccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttc tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattct tttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctg ctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaaggg caatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaac gtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcct gcca SEQ ID N0:36 is the nucleic acid sequence of RD29A-AtCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the AtCPP sense sequence.
SEQ ID N0:37 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca l0 gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatcctcccaatgtccaagctcgtgtgca ataaccgccacaatttcatcctcattcttgcactgctgaatcaacgtatcataaagaac aatccttttgttcttaaagaaaccatacatgtaagcattgctatggcttgaccttgtag atccatcgacaacaaacagcttcttcaaaggaaactttagggaagaagcaagtttctca atcttctcccggaggtctccatctggaagaggagtgaatttgttgaagagcggtgctat caagaccgggtatatagtcatcatcactagagacaggataaacatgaatgcccacagat agatggcaagataaggacctcctttctggactatgaaaattatcgcagcaacaatgggt gggcctagtatgacagagaggaatgttcctttgatcatgtccctaatgaacatccatat tgtttgtttgttgaacccatgccgagactcgatcacgaaagttgagtacaaagaaaatg gcaaatcagtgatctgtgaccatgtcataacaccagccaagaatgaaagagtatgcagt atttcattctccggatcaaggcccaacctcggtaaaagaggatccccATCTACCCGCTT
CGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACC
GTTCTACTTTACTGGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCG
ATAACGTGCTGATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTAC
CGTACCTCGCATTACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCAT
CGTGGTGATTGATGAAACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCG

AAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAG
CAAGCGCACTTACAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGT
GGTGATGTGGAGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATT
TCGCGCCACTGGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTC
AATGTAATGTTCTGCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTG
CCTGAACCGTTATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGG
TACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAA
GAGTATCAGTGTGCATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGT
CGTCGGTGAACAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCG
TTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTT
CTGCTGCAAAAACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAA
ACAATGAATCAACAACTCTCCTGGCGCACCATCGTCGGCTACAGCCTCGGGAATTGCTA
CCGAGCTCttttaccgaggttgggccttgatccggagaatgaaatactgcatactcttt cattcttggctggtgttatgacatggtcacagatcactgatttgccattttctttgtac tcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatgttcat tagggacatgatcaaaggaacattcctctctgtcatactaggcccacccattgttgctg cgataattttcatagtccagaaaggaggtccttatcttgccatctatctgtgggcattc atgtttatcctgtctctagtgatgatgactatatacccggtcttgatagcaccgctctt caacaaattcactcctcttccagatggagacctccgggagaagattgagaaacttgctt cttccctaaagtttcctttgaagaagctgtttgttgtcgatggatctacaaggtcaagc catagcaatgcttacatgtatggtttctttaagaacaaaaggattgttctttatgatac gttgattcagcagtgcaagaatgaggatgaaattgtggcggttattgcacacgagcttg gacattgggagctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaag attgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgtta agcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgatt agagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaacta ggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtc gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccc aacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgc cacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat ttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagt gggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaa tagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttg atttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgg ggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaat cagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtcc gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:37 is the nucleic acid sequence of RD29A-HP-AtCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the AtCPP anti-sense sequence. Upper case sequence represents the truncated GUS fragment. Bold and underlined sequence represents the A.
to thaliana CaaX prenyl protease sense fragment.
SEQ ID N0:38 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg 15 ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg 20 ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct 25 cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca 30 tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatccTTAATCTGTCTTCTTGTCTTCTCC
ATCAGTGGCTCGAAGCCTTTCAACAAGAGGAGGATGTGAGTAGTGATAAGCTGAGTACA
ATGGATCAGTGTTCATTGTTGATAAGTTCTCTTCCTGTAGTTTCACTAGAGCAGGACGA
AGATCTTTTGCATAGTCAAGCTTCACAGCAAAAGCATCAGCCTGAAACTCAAACGCTCG
ACTAACGAGGTTCAGGCCAAAGCTTACTAGATGTTGCAGTGGTATTACAGTGTGCTGAA
ATATGATCAAACCAATGAGAACAGGCTGTGTATCAAATCCGAAACTCCTGAAGAGATCA
GTGGAGTTTCTGAGAAGAGTGTATCCTCCAAATTGTAAGAAGGCAAGGATTTGAACTGC
AATGAACGAGTATGTAGTGTGATTCAGTTTCCAATGTCCAAGCTCGTGTGCAATAACCG
CCACAATTTCATCCTCATTCTTGCACTGCTGAATCAACGTATCATAAAGAACAATCCTT
TTGTTCTTAAAGAAACCATACATGTAAGCATTGCTATGGCTTGACCTTGTAGATCCATC
GACAACAAACAGCTTCTTCAAAGGAAACTTTAGGGAAGAAGCAAGTTTCTCAATCTTCT
CCCGGAGGTCTCCATCTGGAAGAGGAGTGAATTTGTTGAAGAGCGGTGCTATCAAGACC
GGGTATATAGTCATCATCACTAGAGACAGGATAAACATGAATGCCCACAGATAGATGGC
AAGATAAGGACCTCCTTTCTGGACTATGAAAATTATCGCAGCAACAATGGGTGGGCCTA
GTATGACAGAGAGGAATGTTCCTTTGATCATGTCCCTAATGAACATCCATATTGTTTGT
TTGTTGAACCCATGCCGAGACTCGATCACGAAAGTTGAGTACAAAGAAAATGGCAAATC
AGTGATCTGTGACCATGTCATAACACCAGCCAAGAATGAAAGAGTATGCAGTATTTCAT
TCTCCGGATCAAGGCCCAACCTCGGTAAAACAGCTCCAGACATCTTCCAAAACCAAGGC
AAGATCCCAAAGAACAAAATTGCAGAGTCCATAAGTATAGTTACAAACTCATGAACAAA
GTGAAAATAGCTTTTGTCAAGACTGTATGCTCGTGATTTCTCAAACTTCTCTTGGCTAA
TTACACCAACCAAGGTTTTCGGGAGAGTTGGAAGCTTGAGAGCAGTGAGTTGCCTCAGA
TCCAAATACGTCTCAAAAATGTACATCACTATCATAAAACCCACGACGGTTTCCATGAA
AGGAATCGCCATcccctcgaatttccccgatcgttcaaacatttggcaataaagtttct taagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattac gttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttat gattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct tCCCaacagttgCgCagCCtgaatggCgCCCgCtCCtttCgCtttCttCCCttCCtttC
tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattct tttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctg ctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaaggg caatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaac gtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcct gcca SEQ ID N0:38 is the nucleic acid sequence of RD29A-antisense-AtCPP.
to Italicized sequences are the right and left border repeats. Underlined sequence is the RD29A promoter. Sequence in upper case sequence is the AtCPP anti-sense sequence.
SEQ ID N0:39 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga 15 caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa 20 ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa 25 gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac 30 ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagctGGGAAATTTTTCGCCAGTTCTAAATATCCGGAAACC
TCTTGGGATGCCATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAG
GTGGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGATACCTCT
GCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGCACCGTGGAGTAAGAAG
ACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTGATGTAGTATCTCCATTGACGTAAGG
GATGACGCACAATCCAACTATCCATCGCAAGACCATTGCTCTATATAAGAAAGTTAATA
TCATTTCGAGTGGCCACGCTGAGGGGGATCCatggcgattcctttcatggaaaccgtcg tgggttttatgatagtgatgtacatttttgagacgtatttggatctgaggcaactcact gctctcaagcttccaactctcccgaaaaccttggttggtgtaattagccaagagaagtt tgagaaatcacgagcatacagtcttgacaaaagctattttcactttgttcatgagtttg taactatacttatggactctgcaattttgttctttgggatcttgccttggttttggaag atgtctggagctgttttaccgaggttgggccttgatccggagaatgaaatactgcatac tctttcattcttggctggtgttatgacatggtcacagatcactgatttgccattttctt tgtactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatg ttcattagggacatgatcaaaggaacattcctctctgtcatactaggcccacccattgt tgctgcgataattttcatagtccagaaaggaggtccttatcttgccatctatctgtggg cattcatgtttatcctgtctctagtgatgatgactatatacccggtcttgatagcaccg ctcttcaacaaattcactcctcttccagatggagacctccgggagaagattgagaaact tgcttcttccctaaagtttcctttgaagaagctgtttgttgtcgatggatctacaaggt caagccatagcaatgcttacatgtatggtttctttaagaacaaaaggattgttctttat gatacgttgattcagcagtgcaagaatgaggatgaaattgtggcggttattgcacacga gcttggacattggaaactgaatcacactacatactcgttcattgcagttcaaatccttg ccttcttacaatttggaggatacactcttctcagaaactccactgatctcttcaggagt ttcggatttgatacacagcctgttctcattggtttgatcatatttcagcacactgtaat accactgcaacatctagtaagctttggcctgaacctcgttagtcgagcgtttgagtttc aggctgatgcttttgctgtgaagcttgactatgcaaaagatcttcgtcctgctctagtg aaactacaggaagagaacttatcaacaatgaacactgatccattgtactcagcttatca ctactcacatcctcctcttgttgaaaggcttcgagccactgatggagaagacaagaaga cagattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaag attgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgtta agcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgatt agagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaacta ggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtc gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccc aacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgc cacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat ttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagt gggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaa tagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttg atttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgg ggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaat cagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtcc gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:39 is the nucleic acid sequence of MuA-AtCPP. Italicized sequences are the right and left border repeats. Sequence in upper case is the MuA
promoter. The A.

thaliana CaaX prenyl protease sense sequence is in bold.
SEQ ID N0:40 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt l0 caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagctGGGAAATTTTTCGCCAGTTCTAAATATCCGGAAACC
IS TCTTGGGATGCCATTGCCCATCTATCTGTAATTTATTGACGAAATAGACGAAAAGGAAG
GTGGCTCCTATAAAGCACATCATTGCGATAACAGAAAGGCCATTGTTGAAGATACCTCT
GCTGACATTGGTCCCCAAGTGGAAGCACCACCCCATGAGGAGCACCGTGGAGTAAGAAG
ACGTTCGAGCCACGTCGAAAAAGCAAGTGTGTTGATGTAGTATCTCCATTGACGTAAGG
GATGACGCACAATCCAACTATCCATCGCAAGACCATTGCTCTATATAAGAAAGTTAATA
TCATTTCGAGTGGCCACGCTGAGGGGGATCGGGATGGCGTTTCCCTACATGGAAGCCGT
TGTCGGATTTATGATATTAATGTACATTTTTGAAACTTACTTGGATGTGCGACAACATA
GGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAGGGTGTTATCAGCCAAGAGAAA
TTTGAGAAATCTAGAGCCTATAGTCTTGATAAAAGCCACTTCCATTTTGTTCACGAGTT
TGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGGGGTATTGCCCTGGTTTTGGA
AGAAATCAGGAGATTTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCAT
ACCCTTGCCTTCTTAGCAGGGCTGATGATTTGGTCACAGATAACAGATTTGCCCTTTTC
TCTGTACTCAACTTTTGTGATTGAGGCCCGTCATGGTTTTAATAAGCAAACACCATGGT
TATTCTTTAGGGACATGCTTAAAGGAATTTTCCTTTCTGTAATAATTGGTCCACCTATT
GTGGCTGCAATCATTGTAATAGTACAGAAAGGAGGTCCATACTTGGCCATCTATCTTTG
GGTTTTTACGTTTGGTCTTTCTATTGTGATGATGACCCTTTATCCAGTACTAATAGCTC
CACTCTTCAATAAGTTCACTCCACTTCCAGATGGTCAACTCAGGGAGAAAATCGAGAAA
CTTGCTTCCTCCCTCAACTATCCGTTAAAGAAACTATTTGTTGTCGATGGATCCACAAG
ATCAAGTCACAGCAATGCCTATATGTATGGATTCTTCAAGAACAAGAGGATTGTCCCTT
ATGACACATTAATTCAACAGTGCAAAGACGATGAGGAAATTGTTGCTGTTATTGCCCAT

GAGTTGGGACACTGGAAGCTCAACCATACTGTGTACACATTTGTTGCTATGCAGATTCT
TACACTTCTACAATTTGGAGGATATACACTAGTGCGAAATTCAGCTGATCTGTATCGAA
GCTTTGGGTTTGATACGCAGCCAGTCCTCATTGGGCTCATCATATTTCAGCATACTGTA
ATCCCACTTCAGCAATTGGTCAGCTTTGGTCTGAACCTAGTCAGCCGATCATTTGAATT
TCAGGCTGATGGCTTTGCCAAGAAGCTTGGATATGCATCTGGATTACGCGGTGGTCTTG
TGAAACTACAGGAGGAGAATCTGTCAGCTATGAATACAGATCCTTGGTACTCTGCTTAT
CACTATTCTCATCCTCCCCTTGTTGAAAGATTGGCCGCGCTGGACGAACCGGATAAGAA
GGAAGACTAAgagctcgaatttccccgatcgttcaaacatttggcaataaagtttctta agattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgt l0 taagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatga ttagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaac taggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccg tcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgca gcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttc CCaaCagttgCgcagCCtgaatggCgCCCgCtCCtttCgCtttCttCCCttCCtttCtC
gccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccg atttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgta gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttt aatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttt tgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgct ggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggca atcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgt ccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgc ca SEQ ID N0:40 is the nucleic acid sequence of MuA-GmCPP. Italicized sequences are the right and left border repeats. Sequence in upper case is the MuA
promoter. The G. max CaaX prenyl protease sense sequence is in upper case and bold.
SEQ ID N0:41 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact aatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaggatccccgggatggcgtttccctacatggaagccg ttgtcggatttatgatattaatgtacatttttgaaacttacttggatgtgcgacaacat agggccctcaaacttcctactcttccaaagactttagagggtgttatcagccaagagaa atttgagaaatctagagcctatagtcttgataaaagccacttccattttgttcacgagt ttgtgacaatagtgacagactctacaattttgtactttggggtattgccctggttttgg aagaaatcaggagattttatgacaatagctggtttcaatgctgagaatgaaatactgca tacccttgccttcttagcagggctgatgatttggtcacagataacagatttgccctttt ctctgtactcaacttttgtgattgaggcccgtcatggttttaataagcaaacaccatgg ttattctttagggacatgcttaaaggaattttcctttctgtaataattggtccacctat tgtggctgcaatcattgtaatagtacagaaaggaggtccatacttggccatctatcttt gggtttttacgtttggtctttctattgtgatgatgaccctttatccagtactaatagct ccactcttcaataagttcactccacttccagatggtcaactcagggagaaaatcgagaa acttgcttcctccctcaactatccgttaaagaaactatttgttgtcgatggatccacaa gatcaagtcacagcaatgcctatatgtatggattcttcaagaacaagaggattgtccct tatgacacattaattcaacagtgcaaagacgatgaggaaattgttgctgttattgccca tgagttgggacactggaagctcaaccatactgtgtacacatttgttgctatgcagattc ttacacttctacaatttggaggatatacactagtgcgaaattcagctgatctgtatcga agctttgggtttgatacgcagccagtcctcattgggctcatcatatttcagcatactgt aatcccacttcagcaattggtcagctttggtctgaacctagtcagccgatcatttgaat ttcaggctgatggctttgccaagaagcttggatatgcatctggattacgcggtggtctt gtgaaactacaggaggagaatctgtcagctatgaatacagatccttggtactctgctta tcactattctcatcctccccttgttgaaagattggccgcgctggacgaaccggataaga aggaagactaagagctcgaatttccccgatcgttcaaacatttggcaataaagtttctt aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacg l0 ttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatg attagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaa ctaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggcc gtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc agcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccctt cccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttcc gatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt taatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattctt 2o ttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgc tggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggc aatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacg tccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctg cca SEQ ID N0:41 is the nucleic acid sequence of pBI121-GmCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter.
The G. max CaaX prenyl protease sense sequence is in bold.
3o SEQ ID N0:42 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact l0 gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaccggttcgtccagcgcggccaatctttcaacaaggg gaggatgagaatagtgataagcagagtaccaaggatctgtattcatagctgacagattc tcctcctgtagtttcacaagaccaccgcgtaatccagatgcatatccaagcttcttggc aaagccatcagcctgaaattcaaatgatcggctgactaggttcagaccaaagctgacca attgctgaagtgggattacagtatgctgaaatatgatgagcccaatgaggactggctgc gtatcaaacccaaagcttcgatacagatcagctgaatttcgcactagtgtatatcctcc aaattgtagaagtgtaagaatctgcatagcaacaaatgtgtacacagtatggttgagct tccagtgtcccaactcatgggcaataacagcaacaatttcctcatcgtctttgcactgt tgaattaatgtgtcataagggacaatcctcttgttcttgaagaatccatacatataggc attgctgtgacttgatcttgtggatccccATCTACCCGCTTCGCGTCGGCATCCGGTCA
GTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTTACTGGCTT
TGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGCTGATGGTGC
ACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACCCT
TACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGATGAAAC
TGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGA
AAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCG

ATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGGAGTATTGC
CAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGAAG
CAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCTGCGAC
GCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGTTATTACGG
ATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTC
TGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGCATGG
CTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACAGGTATG
GAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAAAG
GGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCTGG
ACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATGAatcaacaactc tcctggcgcaccatcgtcggctacagcctcgggaattgctaccgagctcacaagatcaa gtcacagcaatgcctatatgtatggattcttcaagaacaagaggattgtcccttatgac acattaattcaacagtgcaaagacgatgaggaaattgttgctgttattgcccatgagtt gggacactggaagctcaaccatactgtgtacacatttgttgctatgcagattcttacac ttctacaatttggaggatatacactagtgcgaaattcagctgatctgtatcgaagcttt gggtttgatacgcagccagtcctcattgggctcatcatatttcagcatactgtaatccc acttcagcaattggtcagctttggtctgaacctagtcagccgatcatttgaatttcagg ctgatggctttgccaagaagcttggatatgcatctggattacgcggtggtcttgtgaaa ctacaggaggagaatctgtcagctatgaatacagatccttggtactctgcttatcacta ttctcatcctccccttgttgaaagattggccgcgctggacgaaccgggagctcgaattt ccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtc ttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatg taatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacat ttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgg tgtcatctatgttactagatcgggaattcactggccgtcgttttacaacgtcgtgactg ggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagct ggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaat ggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcccc gtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctc gaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagac ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaa ctggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccg atttcggaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgc ttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcact ggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgttattaagttgt ctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:42 is the nucleic acid sequence of pBI121-HP-GmCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter.
Bold sequence is the antisense prenyl protease fragment of G. max. Bold and underlined sequence is the G. max sense prenyl protease fragment and sequence in upper case is the truncated GUS fragment.
1 o SEQ ID N0:43 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt 15 caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc 20 aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga 25 tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat 30 cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca 1o8 acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagaccLttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcacaaaacccttcctctatataaaaaaattcatttcattta gagagaacacgggggactctagaggatccccgggttagtcttccttcttatccggttcg tccagcgcggccaatctttcaacaaggggaggatgagaatagtgataagcagagtacca aggatctgtattcatagctgacagattctcctcctgtagtttcacaagaccaccgcgta atccagatgcatatccaagcttcttggcaaagccatcagcctgaaattcaaatgatcgg ctgactaggttcagaccaaagctgaccaattgctgaagtgggattacagtatgctgaaa tatgatgagcccaatgaggactggctgcgtatcaaacccaaagcttcgatacagatcag ctgaatttcgcactagtgtatatcctccaaattgtagaagtgtaagaatctgcatagca acaaatgtgtacacagtatggttgagcttccagtgtcccaactcatgggcaataacagc aacaatttcctcatcgtctttgcactgttgaattaatgtgtcataagggacaatcctct tgttcttgaagaatccatacatataggcattgctgtgacttgatcttgtggatccatcg acaacaaatagtttctttaacggatagttgagggaggaagcaagtttctcgattttctc cctgagttgaccatctggaagtggagtgaacttattgaagagtggagctattagtactg gataaagggtcatcatcacaatagaaagaccaaacgtaaaaacccaaagatagatggcc aagtatggacctcctttctgtactattacaatgattgcagccacaataggtggaccaat tattacagaaaggaaaattcctttaagcatgtccctaaagaataaccatggtgtttgct tattaaaaccatgacgggcctcaatcacaaaagttgagtacagagaaaagggcaaatct gttatctgtgaccaaatcatcagccctgctaagaaggcaagggtatgcagtatttcatt ctcagcattgaaaccagctattgtcataaaatctcctgatttcttccaaaaccagggca ataccccaaagtacaaaattgtagagtctgtcactattgtcacaaactcgtgaacaaaa tggaagtggcttttatcaagactataggctctagatttctcaaatttctcttggctgat aacaccctctaaagtctttggaagagtaggaagtttgagggccctatgttgtcgcacat ccaagtaagtttcaaaaatgtacattaatatcataaatccgacaacggcttccatgtag ggaaacgccatgagctcgaatttccccgatcgttcaaacatttggcaataaagtttctt aagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacg ttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatg attagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaa ctaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggcc gtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc agcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccctt cccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttcc gatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt taatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattctt ttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgc tggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggc aatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacg tccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctg cca SEQ ID N0:43 is the nucleic acid sequence of pBI121-antisense-GmCPP
Italicized sequences are the right and left border repeats. Underlined sequence is the 35S
promoter. Sequence in bold is the GmCPP anti-sense sequence.
SEQ ID N0:44 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt l0 catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta qaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag _atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatccccgggatggcgtttccctacatgg aagccgttgtcggatttatgatattaatgtacatttttgaaacttacttggatgtgcga caacatagggccctcaaacttcctactcttccaaagactttagagggtgttatcagcca agagaaatttgagaaatctagagcctatagtcttgataaaagccacttccattttgttc acgagtttgtgacaatagtgacagactctacaattttgtactttggggtattgccctgg ttttggaagaaatcaggagattttatgacaatagctggtttcaatgctgagaatgaaat actgcatacccttgccttcttagcagggctgatgatttggtcacagataacagatttgc ccttttctctgtactcaacttttgtgattgaggcccgtcatggttttaataagcaaaca ccatggttattctttagggacatgcttaaaggaattttcctttctgtaataattggtcc acctattgtggctgcaatcattgtaatagtacagaaaggaggtccatacttggccatct atctttgggtttttacgtttggtctttctattgtgatgatgaccctttatccagtacta atagctccactcttcaataagttcactccacttccagatggtcaactcagggagaaaat cgagaaacttgcttcctccctcaactatccgttaaagaaactatttgttgtcgatggat ccacaagatcaagtcacagcaatgcctatatgtatggattcttcaagaacaagaggatt gtcccttatgacacattaattcaacagtgcaaagacgatgaggaaattgttgctgttat tgcccatgagttgggacactggaagctcaaccatactgtgtacacatttgttgctatgc agattcttacacttctacaatttggaggatatacactagtgcgaaattcagctgatctg tatcgaagctttgggtttgatacgcagccagtcctcattgggctcatcatatttcagca tactgtaatcccacttcagcaattggtcagctttggtctgaacctagtcagccgatcat ttgaatttcaggctgatggctttgccaagaagcttggatatgcatctggattacgcggt ggtcttgtgaaactacaggaggagaatctgtcagctatgaatacagatccttggtactc tgcttatcactattctcatcctccccttgttgaaagattggccgcgctggacgaaccgg ataagaaggaagactaagagctcgaatttccccgatcgttcaaacatttggcaataaag tttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttga attacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtt tttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcg cgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattca ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcg ccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatc gCCCttcccaacagttgcgcagcctgaatggcgcccgctCCtttCClCtttCttCCCttC
ctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttag ggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggt tcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccac gttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggct attcttttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttc gcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtg aagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacatta aaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatat atcctgcca SEQ ID N0:44 is the nucleic acid sequence of pRD29A-GmCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the GmCPP sense sequence.
SEQ ID N0:45 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaccggttcgtccagcgcggccaatctttcaa caaggggaggatgagaatagtgataagcagagtaccaaggatctgtattcatagctgac agattctcctcctgtagtttcacaagaccaccgcgtaatccagatgcatatccaagctt cttggcaaagccatcagcctgaaattcaaatgatcggctgactaggttcagaccaaagc tgaccaattgctgaagtgggattacagtatgctgaaatatgatgagcccaatgaggact ggctgcgtatcaaacccaaagcttcgatacagatcagctgaatttcgcactagtgtata tcctccaaattgtagaagtgtaagaatctgcatagcaacaaatgtgtacacagtatggt tgagcttccagtgtcccaactcatgggcaataacagcaacaatttcctcatcgtctttg cactgttgaattaatgtgtcataagggacaatcctcttgttcttgaagaatccatacat ataggcattgctgtgacttgatcttgtggatccccATCTACCCGCTTCGCGTCGGCATC
CGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTTAC
TGGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGCTGA
TGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCAT
TACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGA
TGAAACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGGGCAACA
AGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTA
CAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGGAG
TATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGG
CGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTC
TGCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGTTA
TTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAAAG
AACTTCTGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGTGT
GCATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACA
GGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACA
AGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAA
CGCTGGACTGGCATGAACTTCGGTGAAAA.ACCGCAGCAGGGAGGCAAACAATGAatcaa CaaCtCtCCtggCgCaCCatCgtCggCtaCagCCtCgggaattgCtaCCgagCtCaCaa aatcaaatcacaacaatacctatatatataQattcttcaaQaacaaQaaQattatccct tatgacacattaattcaacagtgcaaagacgatgaggaaattgttgctgttattc~ccca tgagttgggacactggaagctcaaccatactgtgtacacatttgttgctatgcagattc ttacacttctacaatttggaggatatacactagtgcgaaattcagctgatctgtatcga agctttgggtttgatacgcagccagtcctcattgggctcatcatatttcagcatactgt aatcccacttcagcaattggtcagctttggtctgaacctagtcagccgatcatttgaat ttcaggctgatggctttgccaagaagcttggatatgcatctggattacgcggtggtctt gtgaaactacaggaggagaatctgtcagctatgaatacagatccttggtactctgctta tcactattctcatcctccccttgttgaaagattggccgcgctggacgaaccgggagctc gaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttg ccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataatt aacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaatt atacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgc gcgcggtgtcatctatgttactagatcgggaattcactggccgtcgttttacaacgtcg tgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcg l0 ccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagc ctgaatggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggct ttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacgg cacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctg atagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgt tccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggatt ttgccgatttcggaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtg gaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgt ctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgttatta agttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:45 is the nucleic acid sequence of pRD29A-HP-GmCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the GmCPP antisense sequence, bold and underlined sequence is the GmCPP sense sequence.
SEQ ID N0:46 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatcatgagcggag a attaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacg ttgaaggagccactcagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgttca a 3o aagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccctcgg tat ccaattagagtctcatattcactctcaatccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatg ga ttgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctg a tgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaa ct gcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaa gcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaag tatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaaca t cgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctc gcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatggcgatgcct gcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgcta tc aggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacgg t atcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcga aat gaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcgga atcgttttccgggacgccggctggatgatcctccagcgc,ggggatctcatgctggagttcttcgcccacgggatctct gcg gaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcacaacgccacgatcctgagcgaca atatgatcgggcccggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgagatgcaccgcgatatct tgctgcgttcggatattttcgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaag ttt cttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataatt aacat 1 o gtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaac aa aatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcctgtcaatgctgg c ggcggctctggtggtggttctggtggcggctctgagggtggtggctctgagggtggcggttctgagggtggcggctctg a gggaggcggttccggtggtggctctggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatg accgaaaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgctactgattacggtgctg ctatcgatggtttcattggtgacgtttccggccttgctaatggtaatggtgctactggtgattttgctggctctaattc ccaa atggctcaagtcggtgacggtgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttga atgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcac ga caggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggct ttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc at 2o gattacgccaagcttgcatgcctgca~~~a~ccata~at~caattcaatcaaact~aaatttct~caa~aatctcaaac ac~~a~atctcaaa~ttt~aaa~aaaatttatttcttc~actcaaaacaaacttac~aaattta~~ta~aacttatata ca ttatatt~taatttttt~taacaaaatgtttttattattattata~aattttact~~ttaaattaaaaat~aata~aaa a~~t~
aattaa~a~~a~a~a~~a~~taaacattttcttctattttttcatattttca~~ataaattatt~taaaa~tttacaa~
attt ccattt~actagt~taaat~a~gaatattctcta~taa~atcattatttcatctacttcttttatcttctaccagta~a ~~aat aaacaatattta~ctccttt~taaatacaaattaattttccttcttgacatcattcaattttaattttac~tataaaat aaaa~
atcatacctatta~aac~attaa~~a~aaatacaattc~aat~a~aa~~at~t~cc~ttt~ttataataaaca~ccaca c~ac~taaac~taaaat~accacat~at~~~ccaata~acat~~acc~actactaataata~taa~ttacatttta~~a t~~aataaatatcatacc~acatca~tttt~aaa~aaaa~~~aaaaaaa~aaaaaataaataaaa~atatactacc~
acat~a~ttccaaaaa~caaaaaaaaa~atcaa~cc~acaca~acac~c~ta~a~a~caaaat~acttt~ac~tca caccac~aaaaca~ac~cttcatac~t~tccctttatctctctca~tctctctataaactta~t~agaccctcctct~t ttta ctcacaaatat~caaacta~aaaacaatcatca~~aataaa~~~ttt~attacttctatt~~aaa~gactctagaggat ccccgggttagtcttccttcttatccggttcgtccagcgcggccaatctttcaacaaggggaggatgagaatagtgata agcaga gtaccaaggatctgtattcatagctgacagattctcctcctgtagtttcacaagaccaccgcgtaatccagatgcatat ccaagctt cttggcaaagccatcagcctgaaattcaaatgatcggctgactaggttcagaccaaagctgaccaattgctgaagtggg attaca gtatgctgaaatatgatgagcccaatgaggactggctgcgtatcaaacccaaagcttcgatacagatcagctgaatttc gcacta gtgtatatcctccaaattgtagaagtgtaagaatctgcatagcaacaaatgtgtacacagtatggttgagcttccagtg tcccaact catgggcaataacagcaacaatttcctcatcgtctttgcactgttgaattaatgtgtcataagggacaatcctcttgtt cttgaagaat ccatacatataggcattgctgtgacttgatcttgtggatccatcgacaacaaatagtttctttaacggatagttgaggg aggaagca agtttctcgattttctccctgagttgaccatctggaagtggagtgaacttattgaagagtggagctattagtactggat aaagggtca 4o tcatcacaatagaaagaccaaacgtaaaaacccaaagatagatggccaagtatggacctcctttctgtactattacaat gattgca gccacaataggtggaccaattattacagaaaggaaaattcctttaagcatgtccctaaagaataaccatggtgtttgct tattaaaa ccatgacgggcctcaatcacaaaagttgagtacagagaaaagggcaaatctgttatctgtgaccaaatcatcagccctg ctaag aaggcaagggtatgcagtatttcattctcagcattgaaaccagctattgtcataaaatctcctgatttcttccaaaacc agggcaata ccccaaagtacaaaattgtagagtctgtcactattgtcacaaactcgtgaacaaaatggaagtggcttttatcaagact ataggctc tagatttctcaaatttctcttggctgataacaccctctaaagtctttggaagagtaggaagtttgagggccctatgttg tcgcacatcc aagtaagtttcaaaaatgtacattaatatcataaatccgacaacggcttccatgtagggaaacgccatgagctcgaatt tccccg atcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttct gttga attacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgca atta tacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttac t agatcgggaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc a gcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga atggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggg ggctc cctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggc cat cgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaac aac actcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttc gcct gctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtc tcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttg tt tacaccacaatatatcctgcca SEQ ID N0:46 is the nucleic acid sequence of pRD29A-antisense-GmCPP.
Italicized sequences are the right and left border repeats. Underlined sequence is the RD29A promoter. Sequence in bold is the GmCPP antisense sequence.
SEQ ID N0:47 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat CgCCgCtCCCgattCgCagCgCatCgCCttCtatCgCCttCttgaCgagttCttCtgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact ~aatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggt~gct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcacaaaacccttcctctatataaaaaaattcatttcattta agagaacacgggggactctagaggatccatggcgattcctttcatggaaaccgtcgtt ggttttatgatagtgatgtacgtttttgagacgtatttggatctgaggcaacatactgc tctcaagcttcccactctcccaaagactttggttggagtcattagccaagagaagtttg agaaatctcgagcttacagtcttgacaaaagccattttcactttgttcatgagtttgtt actatacttatggactctgcgattctgttctttgggatcttgccttggttttggaagat atctggcggctttctaccaatggtgggactcgatccagagaatgaaatcctgcacactc tttcattcttggctggtcttatgacatggtcacagatcactgatttgccattttctttg tactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatgtt cattagggacatgatcaaaggaatactcctctctgtcatacctgcccctcctatcgttg ccgcaattattgttatagttcagaaaggaggtccttacctcgccatctatctgtgggca ttcatgtttatcctgtctctagtgatgatgactatataccctgttttgattgcacctct tttcaacaagttcactcctcttcctgatggagacctccgggagaagattgagaaacttg cttcttctctaaagtttcctctgaagaagctgtttgttgtcgatggatctacaaggtca agccatagtaatgcttacatgtatggtttcttcaagaacaaaaggattgttctttatga cacattgattcagcagtgccagaatgagaatgaaattgtggcggttattgcacacgagc tgggacactggaagctgaatcacactacatactcgttcattgctgttcaaatccttgcc ttcttgcaatttggaggatacactcttgtcagaaactccactgatctcttcaggagttt tggttttgatacacaaccagttctcattggtttgatcatatttcagcacactgtaatac cacttcaacacctagtaagctttgacctcaaccttgttagtcgagcgtttgagtttcag gctgatgcttttgcagtgaatcttggttatgcaaaggatctacgtcctgccctagtgaa gctacaggaagagaacttatcagcgatgaacacagacccattgtactcagcttatcact actcacaccctcctcttgtagagaggcttcgagccattgatggagaagacaagaagaca gattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagat tgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaag catgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattag agtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactagg ataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcac atccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgcca cgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattt agtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgat ttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgggg caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatca gctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgc aatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:47 is the nucleic acid sequence of pBI121-BnCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter.
Sequence in bold is the BnCPP antisense sequence.
SEQ ID N0:48 l0 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact 15 gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg 20 gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca 25 gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag 30 cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcattt~
gagagaacacgggggactctagaccagtgtcccagctcgtgtgcaataaccgccacaat ttcattctcattctggcactgctgaatcaatgtgtcataaagaacaatccttttgttct tgaagaaaccatacatgtaagcattactatggcttgaccttgtagatccatcgacaaca aacagcttcttcagaggaaactttagagaagaagcaagtttctcaatcttctcccggag gtctccatcaggaagaggagtgaacttgttgaaaagaggtgcaatcaaaacagggtata tagtcatcatcactagagacaggataaacatgaatgcccacagatagatggcgaggtaa ggacctcctttctgaactataacaataattgcggcaacgataggaggggcaggtatgac agagaggagtattcctttgatcatgtccctaatgaacatccatattgtttgtttgttga acccatgccgagactcgatcacgaaagttgagtacaaagaaaatggcaaatcagtgatc tgtgaccatgtcataagaccagccaagaatgaaagagtgtgcaggatttcattctctgg atcgagtcccaccattggtagaaggatccccATCTACCCGCTTCGCGTCGGCATCCGGT
CAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTTACTGGC
TTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGCTGATGGT
GCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACC
CTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGATGAA
ACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCC
GAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGG
CGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGGAGTATT
GCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGA
AGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCTGCG
ACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGTTATTAC
GGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACT
TCTGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGCAT
GGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACAGGTA
TGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAA
AGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCT
GGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATGAatcaacaac tctcctggcgcaccatcgtcggctacagcctcgggaattgctaccgagctcttctacca atggtgggactcgatccagagaatgaaatcctgcacactctttcattcttggctggtct tatgacatggtcacagatcactgatttgccattttctttgtactcaactttcgtgatcg agtctcggcatgggttcaacaaacaaacaatatggatgttcattagggacatgatcaaa ggaatactcctctctgtcatacctgcccctcctatcgttgccgcaattattgttatagt tcagaaaggaggtccttacctcgccatctatctgtgggcattcatgtttatcctgtctc tagtgatgatgactatataccctgttttgattgcacctcttttcaacaagttcactcct cttcctgatggagacctccgggagaagattgagaaacttgcttcttctctaaagtttcc tctgaagaagctgtttgttgtcgatggatctacaaggtcaagccatagtaatgcttaca tgtatggtttcttcaagaacaaaaggattgttctttatgacacattgattcagcagtgc cagaatgagaatgaaattgtggcggttattgcacacgagctgggacactgggagctcga atttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgcc ggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaa catgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattat acatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgc gcggtgtcatctatgttactagatcgggaattcactggccgtcgttttacaacgtcgtg actgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgcc agctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcct gaatggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggcttt ccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgat agacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttc caaactggaacaacactcaaccctatctcgggctattcttttgatttataagggatttt gccgatttcggaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtgga ccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtct cactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgttattaag ttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:48 is the nucleic acid sequence of pBI121-HP-BnCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the 35S promoter.
Sequence in bold is the BnCPP antisense sequence, bold and underlined sequence is the BnCPP sense fragment and upper case indicates the truncated GUS fragment.
SEQ ID N0:49 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagcccacagatggttagagaggctt acgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaatac cttcccaagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacac agagaaagatatatttctcaagatcagaagtactattccagtatggacgattcaaggct tgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccact gaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaa gactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcg tcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctca gaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcgg attccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggct cctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgac agtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttcc aaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacg cacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttg gagagaacacgggggactctagaggatccttaatctgtcttcttgtcttctccatcaat ggctcgaagcctctctacaagaggagggtgtgagtagtgataagctgagtacaatgggt ctgtgttcatcgctgataagttctcttcctgtagcttcactagggcaggacgtagatcc tttgcataaccaagattcactgcaaaagcatcagcctgaaactcaaacgctcgactaac aaggttgaggtcaaagcttactaggtgttgaagtggtattacagtgtgctgaaatatga tcaaaccaatgagaactggttgtgtatcaaaaccaaaactcctgaagagatcagtggag tttctgacaagagtgtatcctccaaattgcaagaaggcaaggatttgaacagcaatgaa cgagtatgtagtgtgattcagcttccagtgtcccagctcgtgtgcaataaccgccacaa tttcattctcattctggcactgctgaatcaatgtgtcataaagaacaatccttttgttc ttgaagaaaccatacatgtaagcattactatggcttgaccttgtagatccatcgacaac aaacagcttcttcagaggaaactttagagaagaagcaagtttctcaatcttctcccgga ggtctccatcaggaagaggagtgaacttgttgaaaagaggtgcaatcaaaacagggtat atagtcatcatcactagagacaggataaacatgaatgcccacagatagatggcgaggta aggacctcctttctgaactataacaataattgcggcaacgataggaggggcaggtatga cagagaggagtattcctttgatcatgtccctaatgaacatccatattgtttgtttgttg aacccatgccgagactcgatcacgaaagttgagtacaaagaaaatggcaaatcagtgat ctgtgaccatgtcataagaccagccaagaatgaaagagtgtgcaggatttcattctctg gatcgagtcccaccattggtagaaagccgccagatatcttccaaaaccaaggcaagatc ccaaagaacagaatcgcagagtccataagtatagtaacaaactcatgaacaaagtgaaa atggcttttgtcaagactgtaagctcgagatttctcaaacttctcttggctaatgactc caaccaaagtctttgggagagtgggaagcttgagagcagtatgttgcctcagatccaaa tacgtctcaaaaacgtacatcactatcataaaaccaacgacggtttccatgaaaggaat cgccatcccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagat tgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaag catgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattag agtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactagg ataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcac atccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgcca cgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattt agtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgat ttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgggg caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatca gctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgc aatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:49 is the nucleic acid sequence of pBI121-antisense-BnCPP.
Italicized sequences are the right and left border repeats. Underlined sequence is the 35S
promoter. Sequence in bold is the BnCPP antisense sequence.
SEQ ID NO:50 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat CgCCgCtCCCgattCgCagCgCatCgCCttCtatCgCCttCttgaCgagttCttCtgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatccatggcgattcctttcatggaaacc gtcgttggttttatgatagtgatgtacgtttttgagacgtatttggatctgaggcaaca tactgctctcaagcttcccactctcccaaagactttggttggagtcattagccaagaga agtttgagaaatctcgagcttacagtcttgacaaaagccattttcactttgttcatgag tttgttactatacttatggactctgcgattctgttctttgggatcttgccttggttttg gaagatatctggcggctttctaccaatggtgggactcgatccagagaatgaaatcctgc acactctttcattcttggctggtcttatgacatggtcacagatcactgatttgccattt tctttgtactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatg gatgttcattagggacatgatcaaaggaatactcctctctgtcatacctgcccctccta tcgttgccgcaattattgttatagttcagaaaggaggtccttacctcgccatctatctg tgggcattcatgtttatcctgtctctagtgatgatgactatataccctgttttgattgc acctcttttcaacaagttcactcctcttcctgatggagacctccgggagaagattgaga aacttgcttcttctctaaagtttcctctgaagaagctgtttgttgtcgatggatctaca aggtcaagccatagtaatgcttacatgtatggtttcttcaagaacaaaaggattgttct ttatgacacattgattcagcagtgccagaatgagaatgaaattgtggcggttattgcac acgagctgggacactggaagctgaatcacactacatactcgttcattgctgttcaaatc cttgccttcttgcaatttggaggatacactcttgtcagaaactccactgatctcttcag gagttttggttttgatacacaaccagttctcattggtttgatcatatttcagcacactg taataccacttcaacacctagtaagctttgacctcaaccttgttagtcgagcgtttgag tttcaggctgatgcttttgcagtgaatcttggttatgcaaaggatctacgtcctgccct agtgaagctacaggaagagaacttatcagcgatgaacacagacccattgtactcagctt atcactactcacaccctcctcttgtagagaggcttcgagccattgatggagaagacaag aagacagattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttct taagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattac gttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttat gattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg CagCdCatCCCCCtttCgCCdgCtggCgtaatagCgaagaggCCCgCaCCgatCgCCCt tCCCaaCagttgCgCagCCtgaatggCgCCCgCtCCtttCgCtttCttCCCttCCtttC
tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattct tttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctg ctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaaggg caatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaac gtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcct gcca SEQ ID NO:50 is the nucleic acid sequence of pRD29A-BnCPP. Italicized 2o sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the BnCPP sense sequence.
SEQ ID NO:51 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc r ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaccagtgtcccagctcgtgtgcaataaccgc cacaatttcattctcattctggcactgctgaatcaatgtgtcataaagaacaatccttt tgttcttgaagaaaccatacatgtaagcattactatggcttgaccttgtagatccatcg acaacaaacagcttcttcagaggaaactttagagaagaagcaagtttctcaatcttctc ccggaggtctccatcaggaagaggagtgaacttgttgaaaagaggtgcaatcaaaacag ggtatatagtcatcatcactagagacaggataaacatgaatgcccacagatagatggcg aggtaaggacctcctttctgaactataacaataattgcggcaacgataggaggggcagg tatgacagagaggagtattcctttgatcatgtccctaatgaacatccatattgtttgtt tgttgaacccatgccgagactcgatcacgaaagttgagtacaaagaaaatggcaaatca gtgatctgtgaccatgtcataagaccagccaagaatgaaagagtgtgcaggatttcatt ctctggatcgagtcccaccattggtagaaggatccccATCTACCCGCTTCGCGTCGGCA
TCCGGTCAGTGGCAGTGAAGGGCGAACAGTTCCTGATTAACCACAAACCGTTCTACTTT
ACTGGCTTTGGTCGTCATGAAGATGCGGACTTGCGTGGCAAAGGATTCGATAACGTGCT
GATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGC
ATTACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATT
GATGAAACTGCTGCTGTCGGCTTTTCGCTCTCTTTAGGCATTGGTTTCGAAGCGGGCAA
CAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACT
TACAGGCGATTAAAGAGCTGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGG
AGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACT
GGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGT
TCTGCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGT

TATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAA
AGAACTTCTGGCCTGGCAGGAGAAACTGTACACCGACATGTGGAGTGAAGAGTATCAGT
GTGCATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAA
CAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAA
CAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAA
AACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAATGAATC
AACAACTCTCCTGGCGCACCATCGTCGGCTACAGCCTCGGGAATTGCTACCGAGCTCtt ctaccaatggtgggactcgatccagagaatgaaatcctgcacactctttcattcttggc tggtcttatgacatggtcacagatcactgatttgccattttctttgtactcaactttcg tgatcgagtctcggcatgggttcaacaaacaaacaatatggatgttcattagggacatg atcaaaggaatactcctctctgtcatacctgcccctcctatcgttgccgcaattattgt tatagttcagaaaggaggtccttacctcgccatctatctgtgggcattcatgtttatcc tgtctctagtgatgatgactatataccctgttttgattgcacctcttttcaacaagttc actcctcttcctgatggagacctccgggagaagattgagaaacttgcttcttctctaaa gtttcctctgaagaagctgtttgttgtcgatggatctacaaggtcaagccatagtaatg cttacatgtatggtttcttcaagaacaaaaggattgttctttatgacacattgattcag cagtgccagaatgagaatgaaattgtggcggttattgcacacgagctgggacactggga gctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcct gttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaat aattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgc aattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaatta tcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtcgttttacaac gtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccct ttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcg cagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgcc ggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgc cctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactc ttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagg gattttgccgatttcggaaccaccatcaaacaggattttcgcctgctggggcaaaccag cgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgc ccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtccgcaatgtgtt attaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID NO:51 is the nucleic acid sequence of pRD29A-HP-BnCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the promoter. Sequence in bold is the BnCPP antisense sequence, bold and underlined sequence is BnCPP sense fragment and the upper case sequence represents the truncated GUS fragment.
SEQ ID N0:52 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagcttgcatgcctgcagggagccatagatgcaattcaatc aaactgaaatttctgcaagaatctcaaacacggagatctcaaagtttgaaagaaaattt atttcttcgactcaaaacaaacttacgaaatttaggtagaacttatatacattatattg taattttttgtaacaaaatgtttttattattattatagaattttactggttaaattaaa aatgaatagaaaaggtgaattaagaggagagaggaggtaaacattttcttctatttttt catattttcaggataaattattgtaaaagtttacaagatttccatttgactagtgtaaa tgaggaatattctctagtaagatcattatttcatctacttcttttatcttctaccagta gaggaataaacaatatttagctcctttgtaaatacaaattaattttccttcttgacatc attcaattttaattttacgtataaaataaaagatcatacctattagaacgattaaggag aaatacaattcgaatgagaaggatgtgccgtttgttataataaacagccacacgacgta aacgtaaaatgaccacatgatgggccaatagacatggaccgactactaataatagtaag ttacattttaggatggaataaatatcataccgacatcagttttgaaagaaaagggaaaa aaagaaaaaataaataaaagatatactaccgacatgagttccaaaaagcaaaaaaaaag atcaagccgacacagacacgcgtagagagcaaaatgactttgacgtcacaccacgaaaa cagacgcttcatacgtgtccctttatctctctcagtctctctataaacttagtgagacc ctcctctgttttactcacaaatatgcaaactagaaaacaatcatcaggaataaagggtt tgattacttctattggaaaggactctagaggatccttaatctgtcttcttgtcttctcc atcaatggctcgaagcctctctacaagaggagggtgtgagtagtgataagctgagtaca atgggtctgtgttcatcgctgataagttctcttcctgtagcttcactagggcaggacgt agatcctttgcataaccaagattcactgcaaaagcatcagcctgaaactcaaacgctcg actaacaaggttgaggtcaaagcttactaggtgttgaagtggtattacagtgtgctgaa atatgatcaaaccaatgagaactggttgtgtatcaaaaccaaaactcctgaagagatca gtggagtttctgacaagagtgtatcctccaaattgcaagaaggcaaggatttgaacagc aatgaacgagtatgtagtgtgattcagcttccagtgtcccagctcgtgtgcaataaccg ccacaatttcattctcattctggcactgctgaatcaatgtgtcataaagaacaatcctt ttgttcttgaagaaaccatacatgtaagcattactatggcttgaccttgtagatccatc gacaacaaacagcttcttcagaggaaactttagagaagaagcaagtttctcaatcttct cccggaggtctccatcaggaagaggagtgaacttgttgaaaagaggtgcaatcaaaaca gggtatatagtcatcatcactagagacaggataaacatgaatgcccacagatagatggc gaggtaaggacctcctttctgaactataacaataattgcggcaacgataggaggggcag gtatgacagagaggagtattcctttgatcatgtccctaatgaacatccatattgtttgt ttgttgaacccatgccgagactcgatcacgaaagttgagtacaaagaaaatggcaaatc agtgatctgtgaccatgtcataagaccagccaagaatgaaagagtgtgcaggatttcat tctctggatcgagtcccaccattggtagaaagccgccagatatcttccaaaaccaaggc aagatcccaaagaacagaatcgcagagtccataagtatagtaacaaactcatgaacaaa gtgaaaatggcttttgtcaagactgtaagctcgagatttctcaaacttctcttggctaa tgactccaaccaaagtctttgggagagtgggaagcttgagagcagtatgttgcctcaga tccaaatacgtctcaaaaacgtacatcactatcataaaaccaacgacggtttccatgaa aggaatcgccatcccctcgaatttccccgatcgttcaaacatttggcaataaagtttct taagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattac gttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttat gattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttg cagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccct tcccaacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttc tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattct tttgatttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctg ctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaaggg caatcagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaac gtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcct gcca SEQ ID N0:52 is the nucleic acid sequence of pRD29A-antisense-BnCPP.
Italicized sequences are the right and left border repeats. Underlined sequence is the RD29A promoter. Sequence in bold is the BnCPP antisense sequence.
SEQ ID N0:53 gtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacga t0 caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcg ggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagc cgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgtt caaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccact gacgttccataaattcccctcggtatccaattagagtctcatattcactctcaatccaa 15 ataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattgcacgcagg ttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcg gctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtg gctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa 20 gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgac 25 ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgt gatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtat cgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgag cgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatt 30 tcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc ggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacgggatctc tgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcaca acgccacgatcctgagcgacaatatgatcgggcccggcgtccacatcaacggcgtcggc ggcgactgcccaggcaagaccgagatgcaccgcgatatcttgctgcgttcggatatttt cgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttg aattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggt ttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcc tgtcaatgctggcggcggctctggtggtggttctggtggcggctctgagggtggtggct ctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct ggttccggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccga aaatgccgatgaaaacgcgctacagtctgacgctaaaggcaaacttgattctgtcgcta ctgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggt aatggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacgg tgataattcacctttaatgaataatttccgtcaatatttaccttccctccctcaatcgg ttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgc ttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagctgggaaatttttcgccagttctaaatatccggaaacc tcttgggatgccattgcccatctatctgtaatttattgacgaaatagacgaaaaggaag gtggctcctataaagcacatcattgcgataacagaaaggccattgttgaagatacctct gctgacattggtccccaagtggaagcaccaccccatgaggagcaccgtggagtaagaag acgttcgagccacgtcgaaaaagcaagtgtgttgatgtagtatctccattgacgtaagg gatgacgcacaatccaactatccatcgcaagaccattgctctatataagaaagttaata tcatttcgagtggccacgctgagggggatccatggcgattcctttcatggaaaccgtcg ttggttttatgatagtgatgtacgtttttgagacgtatttggatctgaggcaacatact gctctcaagcttcccactctcccaaagactttggttggagtcattagccaagagaagtt tgagaaatctcgagcttacagtcttgacaaaagccattttcactttgttcatgagtttg ttactatacttatggactctgcgattctgttctttgggatcttgccttggttttggaag atatctggcggctttctaccaatggtgggactcgatccagagaatgaaatcctgcacac tctttcattcttggctggtcttatgacatggtcacagatcactgatttgccattttctt tgtactcaactttcgtgatcgagtctcggcatgggttcaacaaacaaacaatatggatg ttcattagggacatgatcaaaggaatactcctctctgtcatacctgcccctcctatcgt tgccgcaattattgttatagttcagaaaggaggtccttacctcgccatctatctgtggg cattcatgtttatcctgtctctagtgatgatgactatataccctgttttgattgcacct cttttcaacaagttcactcctcttcctgatggagacctccgggagaagattgagaaact tgcttcttctctaaagtttcctctgaagaagctgtttgttgtcgatggatctacaaggt caagccatagtaatgcttacatgtatggtttcttcaagaacaaaaggattgttctttat gacacattgattcagcagtgccagaatgagaatgaaattgtggcggttattgcacacga gctgggacactggaagctgaatcacactacatactcgttcattgctgttcaaatccttg ccttcttgcaatttggaggatacactcttgtcagaaactccactgatctcttcaggagt tttggttttgatacacaaccagttctcattggtttgatcatatttcagcacactgtaat accacttcaacacctagtaagctttgacctcaaccttgttagtcgagcgtttgagtttc aggctgatgcttttgcagtgaatcttggttatgcaaaggatctacgtcctgccctagtg aagctacaggaagagaacttatcagcgatgaacacagacccattgtactcagcttatca ctactcacaccctcctcttgtagagaggcttcgagccattgatggagaagacaagaaga cagattaacccctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaag attgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgtta agcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgatt agagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaacta ggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattcactggccgtc gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccc aacagttgcgcagcctgaatggcgcccgctcctttcgctttcttcccttcctttctcgc cacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat ttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagt gggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaa tagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttg atttataagggattttgccgatttcggaaccaccatcaaacaggattttcgcctgctgg ggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaat cagctgttgcccgtctcactggtgaaaagaaaaaccaccccagtacattaaaaacgtcc gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgcca SEQ ID N0:53 is the nucleic acid sequence of MuA-BnCPP. Italicized sequences are the right and left border repeats. Underlined sequence is the MuA
promoter.
Sequence in bold is the BnCPP sense sequence.
Example 5. Southern Analysis Genomic Southern blot analysis of transgenic Arabidopsis was performed using standard techniques known to one skilled in the art. Typically, l Op,g of DNA
was electrophoresed in a 0.8% agarose gel and transferred to an appropriate membrane such as Hybond N+ (Amersham Pharmacia Biotech). Pre-hybridization and hybridization conditions were as suggested by the membrane manufacturer, typically at 65°C. The final stringency wash was typically at 1XSSC and 0.1% SDS at 65°C. The NPTII coding region was typically used as the radiolabeled probe in Southern blot analysis.
Thirty-seven Arabidopsis lines were selected as homozygous pBI121-AtCPP
over-expression lines for further examination. Figure 3 shows a representative blot confirming the presence of the pBI121-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
to Thirty-three Arabidopsis lines were selected as homozygous pBI121-HP-AtCPP
hair-pin down-regulation lines for further examination. Figure 4 shows a representative blot confirming the presence of the pBI l21-HP-AtCPP hair-pin construct. All lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR
assays.
15 Arabidopsis lines were selected as homozygous pRD29A-AtCPP over-expression lines for further examination. Figure 5 shows a representative blot confirming the presence of the pRD29A-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.
Arabidopsis lines were selected as homozygous pRD29A-HP-AtCPP lines for 2o further examination. Figure 6 shows a representative blot confirming the presence of the pRD29A-HP-AtCPP transgene. Lines were confirmed to be transgenic by PCR
analysis using transgene specific primers in the PCR assays.
Example 6: PCR analysis of transgenic plants PCR was used as a method to confirm the presence of the transgene in all 25 transgenic lines and every construct.. Typical PCR mixtures contained: 1X
reaction buffer (IOmM Tris-HCl pH 8.8, l.SmM MgCl2,, SOmM KCI), dNTP's at 200p.M, 1pM
forward and reverse primer, 2.5U. Taq DNA polymerase, and template plus water to a final volume of SOp,L. Reactions were run at 1 minute 94°C, 1 minute 60°C, 1 minute 72°C, for 30 cycles. Primers used in the analysis of pBI121-AtCPP and pBI121-HP-3o AtCPP transgenic plants were as shown in Table 8. Primers used in the analysis of pRD29A-AtCPP were RD29AP1 (SEQ ID N0:66 ) and SEQ ID N0:7. Primers used in the analysis of pRD29A-HP-AtCPP transgenic plants were those identified as (SEQ ID N0:66), SEQ ID N0:8 and SEQ ID N0:8, Nosterm-RV (SEQ ID NO:67 ) Table 8.
pBI121-AtCPP BamFW: 5'-GCCGACAGTGGTCCCAAAGATGG-3' (SEQ ID NO:10) p35S-AtCPP SmaRV: 5'-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3' (SEQ ID N0:7) p35S-HP-AtCPP BamFW: 5'-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3' to (SEQ ID N0:8) p35S-HP-AtCPP SmaRV: 5'-GCAAGACCGGCAACAGGA-3' (SEQ ID N0:13) pRD29AP1: 5'-TTTAAGCTTGGAGCCATAGATGCAATTCAA -3' (SEQ ID N0:66) pRD29AP 1: 5'-TTTAAGCTTGGAGCCATAGATGCAATTCAA -3' (SEQ ID N0:66) Nosterm-RV: 5'-GCAAGACCGGCAACAGGA-3' (SEQ ID N0:67) Example 7: Northern analysis of transgenic plants Total RNA was isolated from developing leaf tissue of 27 35S-AtCPP
Arabidopsis lines (T3 plants). Approximately 10 ~g of total RNA was loaded into each lane. The Northern blot was first probed with P32 labeled, single-stranded antisense transcript of AtCPP which detects sense transcript, then stripped and re-probed with cDNA of ~i-tubulin that was used as a reference. The hybridizing bands of AtCPP and ~i-tubulin were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA), and the ratio of the two hybridizing bands for each sample was obtained.
The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Twenty-one out of twenty-seven lines showed higher expression of AtCPP transcript as compared to the wild type. Values ranged from 104 % to 282 % of wild type. The results of five lines (35, 84, 76, 136, and 156) of the 21 over-expressing lines is shown in Figure 7.
Example 8: Production of polyclonal antibodies against AtCPP
Anti-AtCPP antibodies were generated using AtCPP fusion protein over-expressed in E. coli. The over-expression vector, pMAL-p2, contains 1175 by malE gene that is located upstream of AtCPP and encodes a 43 KDa maltose-binding protein (MBP).
The 1275 by BamHIlSmaI DNA fragment of AtCPP was inserted into pMAL-p2 at BamHI
and SaII sites. The SaII site was converted into blunt end using Klenow fragment. The resulting fusion protein MBP-AtCPP was then over-expressed in DHSa, and purified by to one-step affinity for MBP as described by the manufacturer (New England Biolab). The soluble fraction of the crude bacterial extract containing the MBP-AtCPP
fusion protein was loaded to a amylose column (1.5 cm x 10.0 cm), and the proteins were eluted with 10 mM maltose in column buffer (50 mM Tris-HCI, pH 7.5, 1 mM EDTA, and 200 mM
NaCI). Fractions containing purified MBP-AtCPP fusion protein were pooled, and IS concentrated with a Centriprep-30 concentrator (Amicon). All purification steps were carried out at 4°C. To generate an antibody, the purified fusion protein was further separated by SDS-PAGE and the Coomassie stained band corresponding to the fusion protein was excised. The identity of the fusion protein was confirmed by Western analysis using anti-MBP antibodies (purchased from New England Biolab). The protein 20 was eluted from the gel slice by electroelution and then emulsified in Ribi adjuvant (Ribi Immunochem) to a final volume of 1 ml. MBP-AtCPP protein was injected into a 3 kg New Zealand rabbit on day 1 and booster injections were given on day 21 and day 35 with 175 pg of the protein each time. High-titer antisera were obtained one week after the final inj ection.
25 Example 9: Western blot analysis of 35S-AtCPP transgenic lines using Anti-AtCPP
antibodies.
Western analysis was performed to examine expression level of AtCPP in the transgenic lines compared with that of wild type plants. Anti-Bip antibody, an ER
lumenal protein (Stressgen, Victoria, BC, Canada ) was used as a reference.
Total 3o proteins were extracted from developing leaf tissue of five ABAs lines and a wild type control.. The antigenic protein bands of AtCPP and Bip were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA) and the ratio of the two protein bands for each sample was obtained. The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Data is presented in Figure 7 indicating that the AtCPP protein level was increased in the transgenic lines compared to the wild type plants.
Example 10: ABA sensitivity of transgenic seedlings.
Approximately 100 seeds were assessed per line per 9 cm plate. Seeds were plated on minimal medium (1/2 MS) supplemented with no ABA or 1.0 ~,M ABA.
Plates were chilled for 3 days at 4 °C in the dark, and incubated for up to 21 days at 22 °C with 24 hour continuous light. Plates were assessed for germination, cotyledon expansion, to true leaf development and seedling vigor. Seedlings were assessed for ABA
sensitivity over 21 days of growth at which time sensitive seedlings were arrested at the cotyledon stage, lacked true leaves, and showed inhibition of root growth. Wild type control Columbia plants had two to three pairs of true leaves and a well developed root system.
Lines were categorized as ABA sensitive (ABAs) if less than 1% of plants looked like 15 control, moderately ABA sensitive (ABAMS) if more than 1% but less than 50%
of looked like control, or ABA insensitive (ABAW') if greater than 50% looked like control.
For example, if a plate had 20 healthy seedlings and the control plate had 60 healthy seedlings, the line would be 33% of control and categorized as moderately ABA
sensitive.
20 All four vector constructs (pBI121-AtCPP, pBI121Hp-AtCPP, pRD29AHp-AtCPP, pRD29A-ATCPP) have resulted in transgenic lines of Arabidopsis which have increased sensitivity to ABA which is indicative of stress tolerance. The data for all 4 constructs is shown in Figure 8. Of the lines transformed with the pBI121-AtCPP
construct to over-express the AtCPP gene, 58% (21 out of 36) were classified as 25 sensitive and an added 30% (11 out of 36) were classified as moderately sensitive. These lines were tested again in T4 and TS generations and their ABA sensitivity was still present indicating that ABA sensitivity is an inheritable trait. Of the lines transformed with the pBI121-HP-AtCPP construct to down-regulate the AtCPP gene by double stranded RNA-inhibition, 15% (7 out of 45) were classified as sensitive and 31% (14 out 30 of 45) were classified as moderately sensitive. To illustrate the increased sensitivity of transgenic lines to ABA, Figure 9 shows the results of germination and seedling development over a range of ABA concentrations. Wild type and pRD29A-HP-AtCPP

are compared. Of the lines transformed with pRD29AHp-AtCPP 70% (12 out of 17) showed high sensitivity and 24% (4 out of 17) showed moderate sensitivity to ABA. Of the lines transformed with pRD29A-AtCPP 29% (5 out of 17) showed high sensitivity and 12% (2 out of 17) moderate sensitivity to ABA. Clearly all 4 transgene constructs are altering ABA sensitivity and ABA signal transduction.
Example 11: Drought Experiments Arabidopsis plants were grown five plants per 4" or 3" pot, in a replicated water-stress experiment. All pots were filled with equal amounts of homogeneous premixed and wetted soil. Plants were grown under 16 hour daylight (150-200 ~,mol/m2/s) at 22 °C
l0 and 70% relative humidity. On the day that the first flower opened drought treatment was initiated. First soil water content in each pot was equalized on a weight basis and any further watering of plants was stopped. Daily measurements of soil water content were taken by recording total pot weight. At the end of the drought treatment (6 to 9 days for experiments in 4" pots and 4-5 days for experiments in 3" pots) plants were harvested 15 and shoot dry weights determined. Differences in plant growth were factored into the analysis by expressing water loss on a per gram shoot dry weight basis.
11a) pBI121-AtCPP, Drought stress screen:
Analysis of pBI121-AtCPP transgenic lines during water-stress treatment experiments of up to an eight day period, shows a strong trend towards increased soil 20 water content and reduced water loss per gram of shoot biomass. After three days of water-stress treatment most lines had increased soil water content relative to the wild type control with four out of twenty-four lines, 146, 149, 156 and 97, showing a statistically significant difference. The amount of water lost per gram of shoot biomass was lower for all lines except one (95), and thirteen of these lines were significantly 25 different from the wild type Columbia control (Figure 10). All of the lines showing a statistically significant lower water loss per gram shoot biomass also showed an increased ABA sensitivity. There is also a strong trend, for all but one line (95), which is ABAW' , towards greater shoot biomass at the end of the drought stress treatment. Seven of those lines 136, 146, 23, 46, 76, 84 and 9, were statistically significant from control at 3o a p=0.05 value.

11b) pBIl21-AtCPP, Water loss per gram shoot biomass during water stress treatment:
Lines 35, 76, 95 and a wild type control were grown and placed under a water-stress treatment as above. Plants were harvested at 2 days, 4 days and 6 days of drought treatment. The ABAs lines, 35 and 76, showed a statistically significant reduction in water-loss relative to shoot dry weight at all three time points (Table 9).
Additionally, the two ABAs transgenic lines had increased shoot biomass, due to increased leaf biomass, and maintained higher soil water contents during drought treatment.
Table 9. Water loss (g) per Shoot dry weight (g) after 2, 4 and 6 days of drought-stress treatment. Values in bold indicate statistically significant differences from Columbia.
2 days 4 days 6 days Line Mean Std. ErrorMean Std. ErrorMean Std. Error 35 212.5 3.5 308.0 9.9 297.7 11.2 76 227.2 5.8 321.2 8.5 293.8 5.0 95 287.0 S.1 377.3 14.8 348.5 25.5 Columbia265.3 11.8 408.2 7.7 345.9 6.7 Wild type 11c) pBI121-AtCPP, Drought stress and shoot recovery:
Water-stress tolerance and determination of post drought-treatment recovery ability was assessed using 20 of the 24 pBI121-AtCPP transgenic lines. Drought treatment was imposed for 6 days after which the plants were watered and allowed to grow for 6 days. Recovered shoot fresh biomass was then determined. Soil water content of these plants was measured daily during the drought treatment and the results confirm previously seen trends. All ABA sensitive (ABAs) lines that showed a statistically significantly reduction of water loss on a per gram dry weight basis in experiment 11 a, continued to show a significant greater soil water content than control plants in this experiment (Table 10). Additionally, Table 10 shows that the recovered shoot fresh biomass after 6 days of drought treatment was significantly greater in all the ABAs lines than Columbia.

Tablel0. Soil water content on day 3 of drought treatment and recovered shoot fresh weight after 6 days of drought treatment (values in bold were significantly different from Columbia at p=0.05) ABA statussoil water recovered content shoot day 3 biomass Line ABA Mean (% Std Error Mean (g) Std Error initial) 136 ABA' 46.6 1.9 4.5 0.16 14 ABA' 50.25 0.7 4.1 0.12 146 ABA' 45.9 2.5 4.0 0.11 147 ABA' 45.1 1.7 4.0 0.15 149 ABA' 45.3 1.8 3.8 0.17 156 ABA' 47.1 1.9 4.0 0.134 23 ABA' 49 1.4 4.0 0.17 33 ABA' 46.9 1.6 4.3 0.14 35 ABA' 41.7 1.7 4.0 0.11 46 ABA' 44.8 1.7 3.8 0.09 63 ABA'' 46.3 1.4 4.0 0.19 76 ABA' 47.8 1.0 3.9 0.17 79 ABA' 45.4 1.1 4.1 0.09 84 ABA' 46.8 1.9 4.1 0.16 85 ABA' 45.3 1.9 4.0 0.12 9 ABA' 45.2 2.1 3.9 0.12 93 ABA""' 43.5 1.2 2.8 0.07 94 ABA' 46.9 1.5 3.9 0.13 97 ABA' S3 1.2 3.8 0.16 95 ABAW' 41.9 1.2 2.7 0.06 Columbia ABA ' 41.3 1.0 ~ 2.7 0.04 11d) pBIl21-AtCPP, Seed yield after drought stress treatment:
Seed yield after drought stress during flowering was examined using ten pBI121-AtCPP transgenic lines, eight of which were ABAs. Plants were grown one per 4"
pot and were exposed to 9 days of drought treatment as described above. A second group of plants was grown and maintained under well watered conditions as the optimal group.
After 9 days of drought treatment plants were re-watered and allowed to continue growth and seed set to maturity. After drought-treatment conditions all eight ABAs lines had to increased yields relative to controls, which ranged from 109% to 126% of the Columbia (Table 11). Drought-treatment resulted in a reduction of yield in all lines, including controls, relative to plants grown under optimal conditions. Expression of the seed yields obtained from drought-treated group relative to the same line under optimal conditions shows that the transgenics preserve a larger percentage of optimal seed yield than do is wild type lines.
Table 11. Seed Yield following 9 days drought-treatment ABA statusSeed Yield (g per plant) Line ABA Mean (g) Std Error % Columbia% Optimal 156 ABAJ 0.735 0.044 126.2 83.7 63 ABA 0.675 0.061 116.0 71.0 146 ABA 0.666 0.053 114.4 72.9 94 ABA' 0.644 0.052 110.6 68.8 84 ABA 0.642 0.049 110.4 61.8 76 ABA' 0.631 0.055 108.5 66.6 136 ABA 0.630 0.051 108.3 74.1 35 ABA' 0.614 0.054 105.6 74.2 93 ABA ' 0.567 0.041 97.5 60.0 95 ABAW' 0.388 0.088 66.7 43.4 Columbia ABA""' 0.582 0.060 100 53.8 11e) pBI121-AtCPP, Seed yield and growth under optimal water conditions:
The lines evaluated above and a number of additional lines were examined in a growth and yield experiment under optimal, well-watered conditions. Results indicated that the ABAs lines were shorter at the stage of first open flower, had more rosette leaves, however, by maturity there were no differences in plant height of transgenics and Columbia. Moreover, the ABAs transgenics showed similar or higher seed yields ranging from 95% to 121% of the wild type control (Figure 11).
11g) pRD29A-HP-AtCPP screen for drought tolerant phenotype:
Analysis of 17 transgenic lines identified 7 candidate drought tolerant lines (12, 22, 23, 47, 82, 83, 90) on the basis of higher soil water content and lower water loss per g of shoot dry weight (Tablel2). All 7 drought tolerant candidate lines showed strong ABA
sensitivity and lines that did not show drought tolerance did not show ABA
sensitivity.
Table 12. Soil water content after 3 days of drought treatment and water lost per g shoot dry weight. Values in bold are statistically different from those of Columbia wild type (p=0.05) ABA statussoil water water lost content in 2days/g day 2 shootD
W

Line ABA , Mean (% Std Error Mean (g/g)Std Error initial) 10 ABA 33.4 1.6 199.1 4.5 11 ABAJ 34.6 3.3 173.1 1.6 12 ABAJ 36.2 2.0 179.5 5.0 126 ABA'"'J 32.5 2.6 199.1 4.1 127 ABA 33.5 2.0 195.6 10.6 14 ABA' 32.7 1.2 203 4.9 17 ABA 29.9 1.8 200.7 7.3 22 ABAJ 39.3 2.1 170.0 3.0 23 ABA' 35.7 1.4 174.9 2.6 42 ABA'"" 28 0.7 185.4 5.8 47 ABAJ 35.9 2.2 181.2 7.7 7 ABA"'' 35 1.3 201.8 5.1 82 ABAJ 36.7 2.2 178.3 4.0 83 ABA 40 1.4 180.7 6.9 9 ABA'' 31.4 1.4 173.8 8.7 90 ABA 38.2 1.3 177.6 6.2 93 ABAW' 30.7 1.8 175.3 4.6 Columbia ABA"'' 32.1 1.2 196.9 6.2 Example 12. Growth Analysis The growth analysis of most promising constructs has been set up at 3 stages.
Eight plants per line were grown in 3" pots with one plant per pot at 22C, l6hr light (150-200 ~mol/m2/s) and 70% RH. Plants were harvested at vegetative growth stage (2 week old seedlings), bolting growth stage (at first open flower) and mid-flowering growth stage (5 to 7 days from first open flower). Also, in some growth experiments additional group of plants was grown in 4" pots (one per pot and 10 plants per line) to maturity for seed yield determinations.
l0 12a) pBI121-AtCPP growth under optimal and biotic stress conditions The growth and productivity of pBI121-AtCPP transgenic Arabidopsis lines was examined at several stages of development under optimal growth conditions.
Although optimal growth conditions were maintained, plants were assessed to be under a degree of stress that was later determined to be a result of the soil properties. Soil analysis found a fungal contaminant that was believed to be responsible for the biotic stress.
This stress could be negated by sterilization of the soil prior to use. Eight ABAs lines, two with normal ABA sensitivity (ABAW') and a wild type Columbia control were analyzed.
Figure 12 presents the results of various growth (from mid-flowering stage) and yield parameters and each trait is expressed as a percentage of the Columbia control.
The results strongly support an enhanced growth phenotype. This enhanced growth phenotype is present at all growth stages. At the vegetative stage, all ABAs transgenic plants showed an increase in leaf number relative to that of the wild type with four of the l0 eight lines showing a statistically significant difference. The two ABAW' lines showed the same or fewer leaves relative to wild type.
At the bolting stage ABAs transgenics showed an increase in leaf number but plants were shorter at this stage (first open flower) than controls. The shoot fresh weight of transgenics was significantly increased relative to that of controls, ranging from 80%
15 to 342% of the wild type. The ABAs transgenics displayed a delay in flowering from one to three days. The ABAW' transgenics did not show delayed flowering, increased shoot fresh weight or increased height.
At the flowering stage of development the enhanced growth phenotype is maintained (greater leaf number and fresh weight), however, there were no observable 2o differences in plant height indicating that transgenics bolt shorter but reach same final plant height.
Of particular significance is the observation, that under these conditions (biotic stress due to presence of fungi in the soil) yields of the ABAs transgenics were significantly higher, ranging from 120% to 229% of the wild type control. The ABAW' 25 lines showed similar or slightly reduced yields relative to the Columbia control. This finding indicates that ABAs transgenic lines are affected less by the biotic stress. This observation has been confirmed, where 5 of the drought tolerant lines were grown in contaminated soil to maturity. The seed yields of transgenic lines, even though greatly reduced relative to optimal conditions, were 2.5 to 4.5 fold higher than those of 3o Columbia wild type (Table 13).
Table 13. Seed yield of pBIl21-AtCPP lines grown in contaminated soil. Values in bold indicate statistical differences at p=0.05 Line ABA Seed Yield per plant% of Columbia sensitivity(g) 156 ABA 0.33 0.04 316%

23 ABA 0.35 0.05 336%

76 ABA 0.31 0.04 296%

84 ABA 0.25 0.33 237%

9 ABA 0.48 0.05 455%

ColumbiaABAW' 0.11 0.03 12b) pBI121-AtCPP early seedling growth:
Four ABAs and one ABAW'line plus Columbia were examined for early seedling growth on agar plates. Twenty seeds were plated in a line on agar plates containing 50%
MS with 1 % sucrose and vitamins and 6 plates per line were used. Plates were placed on slants, which allowed roots to grow downwards. Root length was measured on 7-day old seedlings and shoot and root biomass determined on 11-day old seedlings. Two of the ABAs transgenic lines had significantly longer roots and all 4 ABAs lines had shoot dry 1o weights 114% to 123% of controls and root dry weights of 116% to 151% of controls.As a result, the shoot biomass to rootbiomass ratios were slightly reduced in transgenics.
These results indicate that enhanced growth of these transgenics is evident in the early growth stage, shortly after germination, and the root growth is more enhanced relative to shoot growth. In a different experiment seedlings were pulled out of agar and roots were 15 stained with toluidine blue to show their structure. Figure 13 shows that transgenic lines had more extensive lateral root system, which would account for greater root biomass.
12c) pRD29A-HP-AtCPP optimal growth characteristics An optimal growth study has been conducted with 10 lines as described before.
Vegetative growth data showed that two of the lines (12 and 9) had significantly more 20 leaves and seven of the lines (12, 22, 23, 47, 82, 9) had significantly greater shoot biomass. Bolting data showed that eight of the lines (12, 22, 23, 47, 82, 9, 90, 93) were significantly delayed in flowering by one to two days, and seven of the lines were significantly shorter than Columbia at first open flower. All of the lines except 42 and 7 had significantly greater number of rosette leaves and shoot FW and this trend is maintained into the mid-flowering harvest (Figure 14). The plant height, however, by mid-flowering harvest was not significantly different between the transgenic lines and control. All the lines that showed this enhanced growth also showed drought tolerance and ABA sensitivity.
Example 13. Ultrastructure pBI121-AtCPP
Two of the drought tolerant and ABAs lines (35 and 76) plus Wt Columbia were used to examine stem and root cross-sections for any differences in ultrastructure. Free hand sections of mature stems (plants flowering for l Odays) were obtained from above to the first node, stained with toluidine blue and preserved with glycerol.
The stems of transgenic plants appeared to have more dense cellular structure and contain one or two more vascular bundles than those of Columbia Wt indicating more enhanced water and nutrient transport system.
Leaf disks were taken and fresh weights determined. Transgenic leaf disks were significantly heavier, 20-24% greater than corresponding wild type controls.
This increase is believed to be as a result of a thicker leaf.
Example 14. Cold stress experiment pBI121-AtCPP
Four drought tolerant, ABAs lines (156, 23, 35, 76) and one ABAW'(95) line plus wild type Columbia were included in a cold stress study. Plants were grown in 3" pots one per pot) with 10 replicate pots per line at 22C for 10 days (7 days on agar plates and 4 in soil). The cold stress group was moved into 7°C for 5 days while the optimal group was left at 22C. After 5 days in the cold both cold stress group and the optimal group were harvested for shoot biomass determination. ABAs and drought tolerant lines had significantly greater shoot biomass than Columbia in both optimal (25 to 39%
greater shoot fresh weight) and cold stress groups (18 to 44% greater shoot DW) (Table 14).
Results of an eight-day cold stress showed that differences between the transgenic lines and Columbia were even more pronounced (53 to 61% greater shoot fresh weight).
This result indicates greater plant vigor and better ability of transgenics to cope with cold stress.
Table 14. Shoot fresh weight of optimal and cold stressed (SC for Sd) pBI121-AtCPP.
Values in bold indicate statistical difference at p=0.05 Line ABA Optimal Cold sensitivityshoot stress FW shoot FW

mg % of Columbiamg % of Columbia 156 ABA 95.4 137% 23.1 118%
3.7 0.7 23 ABA 96.3 139% 28.3 144%
3.9 1.5 35 ABAJ 87.0 125% 25.3 130%
1.7 1.4 76 ABA 94.7 136% 27.3 140%
2.2 1.5 95 ABAWt 67 2.4 96% 21.4 109%
1.0 ColumbiaABAWt 69 1.9 19.6 1.1 Example 15. Drought stress under high temperature pBI121-AtCPP
A drought stress experiment was conducted as described above except that day temperature of 32°C (l6hr) and night temperature of 22°C (8hr) was maintained. These temperatures were achieved daily over a 2hr ramping period. Four ABAS and one ABAW~Iine plus Columbia were included. Plants were monitored daily for water loss and soil water content and after 5 days of drought treatment half of the plants were harvested and the other half was re-watered and allowed to recover for four days. Shoots were harvested and shoot fresh weight determined. The results (Table 15) of this experiment 1 o showed that previously identified drought tolerant lines maintained their drought tolerant phenotype at high temperature and were able to recover well from the drought stress at high temperature Table 15. Soil water content on day 2 and water lost in 2 days/final shoot dry weight 15 plus recovery shoot FW after Sdays of drought stress at 32C day and 22C
night temperatures. Values in bold indicate significant differences from the Columbia control.
line ABA soil water water lost in recovered shoot sensitivitycontent day 2d/shoot DW FW (g) 136 ABA 50.4 1.1 485.7 18.5 1.30 0.04 146 ABA 52.1 1.0 504.5 7.9 1.15 0.04 35 ABA 52.2 0.8 502.8 15.8 1.19 0.02 76 ABA 52.1 0.6 435.6 10.5 1.11 0.03 95 ABAWt 50.0 0.9 518.2 13.0 0.86 0.03 ColumbiaABAWt 48.6 0.6 559.7 19.0 0.84 0.03 Example 16. Heat stress and seed yield pBI121-AtCPP
Two ABAs lines and one ABAW'line plus Columbia were examined for the effect of heat stress during flowering on the final seed yield. Plants were grown in 4 inch pots (one/pot) as described above and 9 days from first open flower the temperature was ramped from 22 C to 43C over 2 hours and plants were kept at 43C for 2hr.
Temperature was then ramped back to 22C over 2 hours and plants were grown under optimal conditions until maturity. The seed yields from this experiment are shown in Table 16.
One of the drought tolerant lines (35) had significantly greater yield than Columbia.
l0 Table 16. Seed yield of pBI121-AtCPP lines after two hour 43C heat stress 9 days from first open flower. Values in bold are statistically significant from Columbia.
line ABA seed yield (g/plant)seed yield (% of col.) sensitivity 35 ABAJ 0.55 0.05 347%

76 ABA 0.24 0.03 148%

95 ABAWt 0.11 0.02 69%

ColumbiaABAWt 0.16 0.03 The effect of heat shock on lines of pBI121-AtCPP at the early flowering stage was assessed. Three ABAs lines (76, 136, 97) a ABAW'line (95) and a Columbia wild type control were seeded in 128 cell flats, one flat per line. At the early flowering stage flats were exposed to a temperature of 46.8°C for 50 minutes and then returned to normal growth conditions. Lack of continued growth from main meristems was defined as main meristem death and scored for each line. Data is shown in Table 17.
Table 17. Meristem death due to heat shock Line Wt 95 76 136 97 Death 91 97 79 59 18 Example 17. Stomata density determinations pBI121AtCPP
Two ABAs lines (76 and 35) plus Columbia were examined for stomata density on the upper and lower leaf surface. Nail polish imprints of the upper and lower epidermis were obtained from a fully expanded leaf #5. These imprints were analyzed under the microscope and the number of stomata per 8.7 x 10~g m2 were counted.
There were no significant differences found between transgenics and Columbia in the stomata of the upper or lower epidermis (Table 18). The increases seen in drought tolerance and reduced water loss is not attributable to a reduced number of leaf stomata.
to Table 18. Stomata numbers per 8.7 x 10-8 m2 of abaxial and adaxial epidermis of fully expanded leaf #5 in pBI121AtCPP.
line ABA sensitivitystomata on upperstomata on lower epidermis epidermis Columbia ABAWt 57 6 116 11 Example 18. CPP Consensus Sequences Also included in the invention is the CPP consensus sequences. The consensus sequences were generated by alignment of the CPP polypeptide and nucleic acid ssequences as well as sequences homogous using the program BioEdit.
The "x" in the consensus sequence represents any amino acid or nucleotide.
Preferably "x" a conservative amino acid or nucleotide substitution. More preferably, "x" is the most amino acid or nucleotide most prevalent at a given postion.
For example, the amino acid at postion 145 of SEQ ID NO: 73 is a proline as it occurs 66%
of the time.
Table 19. ClustalW Analysis of BASF Nucleic Acids 1) BASF_ATl (SEQ ID N0:21) 2) BASF_AT2 (SEQ ID N0:23) 3) BASF-Corn (SEQ ID N0:25) 4) aasF-soy (SEQ ID N0:27) 5) Consensus (SEQ ID N0:68) ....I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ 1 BASF_AT2 ______-_________________________________________________-___ 1 BASF-Corn ____________________________________________________________ 1 BASF-Soy CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGTACGCGGGGGGAGACG 60 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 60 ....1....I....1....I....1....1....1....1....1....1....1....1 HASF_AT1 __________________________________________-_________________ 1 BASF_AT2 ____________________________________________________________ 1 HASF-Corn ____________________________________________________________ 1 BASF-Soy CATGGTTCTGAACTAATTGTTATAAATAATACCTAAAATTTTGAGTTGTCCTAAACATTG 120 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 120 ....I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ 1 BASF_AT2 ____________________________________________________________ 1 BASF-Corn ____________________________________________________________ 1 BASF-Soy GGGTTTAAACAAATCCAATCTCTCAATATAAAACCCAATGATCTCACCCTCACTCCGTTT 180 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 180 ....1....I....I....1....1....1....1....1....1..../.. .I.. .I
BASF_AT1 ____________________________________________________ BASF AT2 -________________________-__________________________. 8 BASF-Corn ____________________________________________________________ 1 HASF-Soy CTGATTTCTCACTCTTCGTTTCTCGTTCGGTTCATCAGCGTGTGTCTCAGCC~T~ 290 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 290 .I.. .1.. .I.. .I.. .I.. .1.. .1.. .1.. .1.. .1.. .I.. .I
BASF AT1 ~T~TC~~T~ ~~~T~ 68 BASF AT2 ~T~T ~ Cm T T~ 68 BASF-Corn ____________________________________________________________ 1 HASF-Soy ~C~G~TmCmA~T~ ~ ~~ T~C~ 300 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 300 .1.. .I.. .I.. .I.. .I.. .1.. .I.. .I.. .I.. .1.. .1.. .I
BASF_ATl ~C~~TC~CT~T~C~GC~~TT~ 128 HASF_AT2 C TC~CT~T CmG C~ TT~ 128 BASF-Corn ____________________________________________________________ 1 BASF-Soy ~GmC~A~AT~GG~C~A~T~T~G~T~A~AA~ 360 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 360 .I.. .1.. .I.. .1.. .1.. .I.. .1.. .I.. .1.. .I.. .I.. .I
BASF AT1 ~~~T ~C C C T T T 188 BASF AT2 ~TC ~A ~C~C~T~T~TT~ 188 HASF-Corn ____________________-_______________________________________ 1 BASF-Soy TmC TA~C~T~T~C~C~C~ 920 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 920 .I....1....I....1....I....i....l....i....l....l....l....l HASF_ATl C~T~C~~T~C~ 298 BASF_AT2 C T ~ T~ ~C T ~ C~ 298 BASE-Corn __________-_________________________________________________ 1 BASF-Soy T~C~GmA~G~GmC ~G~A~ 480 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 980 BASF_ATl T T T CT T C G~GGTT C T ~ C 308 BASF_AT2 ~T ~TG~T~CAG~T~AC~C~GGTT C T ~ C 308 BASF-Corn ________________________________________AC GAG T ~~ ~~ 20 BASF-Soy ~C ~~ ~ ~ TT~GA~TAGCT TT C ~ T~ 590 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX X XX ~ 590 ....I....I....I....I....I....1....1....1....1....1....1....1 ....i....I....I....1....I....i....l....l....l....l....l....l BASF ATl ~ ~C ~C~ C~ T ~ 368 BASF AT2 ~ T T ~ ~C ' C T ~ 368 BASF-Corn C~ C TCC~ GTT ~ T~ 80 BASF-Soy ~ C C GC~ ~ T 600 Consensus XXX~ XX 600 .I....I....I....I....I....1....1....1....1....1....1....1 BASE_AT1 T C ~ C ~ T G C~' 928 BASF_AT2 T C ~ C ~ T C~~ 928 BASF-Corn C C T T T T T~~ ~~~ 140 BASF-Soy C C ~ T ~ T ~ C T ~ T T~~T 660 Consensus X ~~ 660 .I....I....I....1....1....1....1....1....1....1....1....1 HASF_AT1 ~ C C C~ C 488 BASF_AT2 ~ C C T C~ ~C 988 BASF-Corn ~ T~ C C ~ ~T~ ' ~ T C T G~ 200 BASF-Soy ~CC~ T T C T~~ ~~TT T C T 720 Consensus XX ~ ~ S ~ C C ~ ~ X 720 .I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 C.. C~ T C C ~ ~T T 548 BASF_AT2 C C~ T C ~ ~T T 598 BASF-Corn ~ C T' C~ C ~TT ' ~C T~ 260 BASF-Soy T ~ T~ ~ C~ G ~ ~ CT 780 Consensus X ~ XXX~ ~ C ~ ~ CX ~ C C X 780 ....I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 C C' C ~ T CT 608 BASF_AT2 C C' C ~ T~ CT 608 BASF-Corn C GT T~ G~AT ~C ~ ~ ~ C T 320 BASF-Soy T TT T~C GGT T T ~ CC T T C 840 Consensus X ~ X ~ XXX X X ' ~ ~ X X X 890 .I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT2 C ~ G~ 668 BASF-Corn T~ T ~ T A 380 HASF-Soy T~ ~~T~~ ~ TC 900 Consensus X ~ 1~~',', C C C XXX C C ~ 900 .I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 T~~ T 728 BASF_AT2 T ~ T 728 BASF-Corn C C~~ T C 940 BASF-Soy C ~ ~~~ C C C~~C 960 Consensus X ~ ~~ X C C C~~X~ X X C X X ~ 960 .I....I....I....I....i....1....1....1....1....1....1....) BASF_AT1 T T~~ 788 1' BASF_AT2 ~ G ~T' T ~ T~~ 788 BASF-Corn ~ C~ ~C~ T~~ C ~ T C~~ ~~GC C~ 500 BASF-Soy C~ ~ T ~C~ C ~T~ C~~ 1020 Consensus X~ ~ X ~ 1020 .I.. .1.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
BASF_AT1 T.. ,~T~ G 898 BASF_AT2 T 'T~ G 898 BASE-Corn C ~ ~C~ T~GC~~ ~ ~ ~ ~ TT T ~ 560 BASF-Soy C ~C~ ~C 'T ~ ~'~ T T ~ 1080 Consensus X ~ XX~ X 1080 .I.. .I.. .1....I.. .I.. .I.. .I.. .1.. .I.. .1.. .I.. .I
BASF_AT1 ,.C ~ T C ~ C . C . ~ T 908 HASF_AT2 ~C ' ~T ~C' C C T ~ 908 BASF-Corn ~ ~T ~ ~C ~~~ C~~ ~T~ GTC ~T C T C GC 620 HASF-Soy C ~T ' ~C C~~C ~T~ GT ~ T 1190 Consensus X ~ ~ X XX ~ X X X~f ~~ 1140 HASF_AT1 968 BASF_AT2 968 BASF-Corn 680 BASF-Soy 1200 Consensus 1200 .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .1....1 HASF_AT1 C. . T T. 1028 1' HASF_AT2 C T 1028 r r BASF-Corn ~ T C C ~GGAC ~ ~ ~ T C C ~ ~ C 790 BASF-Soy ~ C T C ~ GC C~ ~ ~ ~T~ 1260 Consensus X~ X~ XXCX ~X ~ X X 1260 BASF_AT1 1088 BASF_AT2 1088 BASF-Corn 800 BASF-Soy 1320 Consensus 1320 BASF_AT2 1198 BASF-Corn 860 BASF-Soy 1380 Consensus 1380 .I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 1208 HASF_AT2 1208 BASF-Corn 920 BASF-Soy 1434 Consensus 1440 BASF_AT1 1268 BASF_AT2 1268 BASF-Corn 980 BASF-Soy ____________________________________________________________ 1434 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1500 .I....I....I....I....I....1....1....1....1....1....1....1 BASF AT1 .~TT~ ____________________________-_______________________ 1275 BASF AT2 ~-___________________-______________________________-_ 1275 BASF-Corn TTAGTCGATCCTTGTATGAGGTTTACATATGGATTTTTCCCTGCCACATGCACA 1040 BASF-Soy ______________________________________________-_____________ 1439 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1560 BASF_AT1 ______________-_____________________________________________ 1275 BASF AT2 ____________________________________________________________ 1275 BASF-Corn CCGATTCAGTGCTTGGATGGTGAGGGTTTTGACATAGGAGTGTTGTCAAAGCTTTAGAGT 1100 BASF-Soy ____________________________________________________________ 1439 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1620 BASF_AT1 ____________________________________________________________ 1275 BASF AT2 ____________________________________________________________ 1275 BASF-Corn GCATCTTTCGGTCAGGTGCAACAGCCTTTCGGTCATTGAGACATATAAGCGAATTAGCTA 1160 BASF-Soy ____________________________________________________________ 1434 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1680 ....I....1....1....1....I....1....1....1....1....1....1....1 BASF AT1 ____________________________________________________________ 1275 HASF AT2 ____________________________________________________________ 1275 T T
1' T T T

T T T T

TC T~T~ ~ CT G
,~, GTGC

X ' X ~ X XXXXXX XXXXX

BASF-CornTTAAAAAAAACAGAACTGTTGCATCAAAAAAAAAAAAAAAAAAGAAACAAAAAAAAAAAA

HASF-Soy _______________________-____________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....I....I....I....I....I....1....1....1....
BASF ___________________________....1....1....1 AT1 ___-_____________________________ _ _______-______-_____________________________________________ _ F1AAAAAAAAAAAG GTGCTCTGCGTTGTTACCACTGCTTG 1280 BASF-Corn HASF-Soy ____________________________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1....1....1....1.
BASF _____________________ _ _____________________ _ CCCTATAGTGATCGTATCAGA
BASF-Corn1301 BASF-Soy _____________________ ConsensusXXXXXXXXXXXXXXXXXXXXX

Table 20. ClustalW Analysis of BASF Amino Acids I) BASF_ATl (SEQ ID N0:22) 2) BASF_AT2 (SEQ ID N0:24) 3) sASF-corn (SEQ ID N0:26) 4) BASF-soy (SEQ ID N0:28) 5) consensus (SEQ ID N0:69) .I....I....I....I....I....1....1....1....1....1....1....1 BASF ATl ~I~~~LT~ ~ 60 BASF AT2 ~I'~ ~L' LT~ ~ 60 BASF-Corn ____________________________________________________________ 1 BASF-Soy ~F~Y~JmHR~E ~ 60 Consensus HASF XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 60 .1.. .I ...I...I.. .. .1 BASF AT1 .I.. P .I 120 HASF_AT2 .1.. D , ~ 120 .1.. P S
.I..
.I..
.I..
VPRL
~
I
VLPR

BASF-Corn _____________________.______________T IE

S

BASF-Soy H~TmT~Y~I(mDFMTI FN ~ I 120 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXX

BASF

BASF

_ 180 BASF

_ 89 BASF-Corn BASF-Soy 180 Consensus 180 BASF

_ 290 BASF

_ 194 BASF-Corn BASF-Soy 240 Consensus 290 BASF

_ 300 HASF

_ 204 BASF-Corn BASF-Soy 300 Consensus 300 BASF

.1.. .1.. .I..
BASF AT1 .I.. ~ .I.. 360 .1.. I' .I..
.I.. .I
.1.. ' .I.. ,P~
.I..
t ~
~

BASF_AT2 360 BASF-Corn 264 BASF-Soy 360 Consensus BASF 360 HASF_AT1 420 BASF_AT2 420 BASF-Corn 329 BASF-Soy 400 Consensus BASF 420 .I....I....I....I....1....1....1....1....1....1....1....1 BASF AT1 ~~p________________________________________________________ q24 BASF_AT2 D _______________________________________________________ 429 BASF-Corn EDSILVGLHMDFSLPHAHRFSAWMVRVLTECCQSFRVHLSVRCNSLSVIETYKRISY 384 BASF-Soy ____________________________________________________________ 900 Consensus HASF XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1....1....I....1....1....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ q24 BASF_AT2 ____________________________________________________________ 424 BASF-Corn KKQNCCIKKKKKKETKKKKKKKKKKKKKKKVLCVVTTACPIVIVS--------------- 429 BASF-Soy ____________________________________________________________ q00 Consensus BASF XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX---------------....I....I....1....1....1....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ q24 BASF_AT2 ____________________________________________________________ q29 BASF-Corn ____________________________________________________________ q29 BASF-Soy ____________________________________________________________ q00 Consensus BASF ____________________________________________________________ ....I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 _________________-__________________________________________ 424 BASF_AT2 ____________________________________________________________ q24 BASF-Corn ____________________________________________________________ q29 BASF-Soy ____________________________________________________________ q00 Consensus BASF ____________________________________________________________ ....1....1....1....I....1....1....1....1....1....1....1....1 HASF AT1 ____________________________________________________________ q29 BASF_AT2 __________________________________-_________________________ q29 BASF-Corn ____________________________________________________________ q29 BASF-Soy ____________________________________________________________ q00 Consensus HASF ____________________________________________________________ ....I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ q29 BASF_AT2 ____________________________________________________________ q29 BASF-Corn ____________________________________________________________ q29 HASF-Soy ____________________________________________________________ q00 Consensus BASF ____________________________________________________________ ....I....I....1....1....I....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ 429 BASF_AT2 ____________________________________________________________ 429 BASF-Corn ____________________________________________________________ 429 BASF-Soy ____________________________________________________________ q00 Consensus BASF ____________________________________________________________ ....1....I....1....1....1....1....1....1....1....1....1....1 BASF_AT1 ____________________________________________________________ 429 BASF_AT2 ____________________________________________________________ q29 BASF-Corn ____________________________________________________________ q29 BASF-Soy ____________________________________________________________ q00 Consensus HASF -___________________________________________________________ ... .I....1....I....1....1....1....1....1........1....1....1 AT1 ___ _________________________________________________________ BASF q29 _ _________________________________________________________ AT2 ___ q24 BASF

_ _________________________________________________________ BASE-Corn ___ q29 BASF-Soy ___ _________________________________________________________ q00 Consensus BASF_________________________________________________________ ___ 525 ... .1....1....1....1....1....1....1.... .... .1....1 AT1 ___ ________________________________.... ... _______ BASF ___-______________929 _ _____________________-___________________________________ AT2 ___ q24 BASF

_ _________________________________________________________ BASF-Corn ___ q29 BASF-Soy ___ _________________________________________________________ q00 Consensus BASF_________________________________________________________ ___ 525 ... .I....I....1....1....1....1....1....1....1....1....1....1 AT1 ___ _________________________________________________________ BASF q24 _ _________________________________________________________ AT2 ___ q24 BASF

_ _________________________________________________________ BASF-Corn ___ 429 BASF-Soy ___ _________________________________________________________ qp0 Consensus BASF_________________-_______________________________________ ___ 525 ... .I....I....I....I....I....1....1....1....1....1....1....1 AT1 ___ ________________________________________________-________ BASF q29 _ _________________________________________________________ AT2 ___ q29 BASF

_ _________________________________________________________ BASF-Corn ___ q29 BASF-Soy ___ _________________________________________________________ Consensus BASF_________________________________________________________ ___ 525 ... .I....I....i....I....I....1....1....1....1....1....1....1 AT1 ___ _________________________________________________________ BASF q24 _ _________________________________________________________ AT2 ___ 924 HASF

_ _________________________________________________________ BASF-Corn ___ q29 BASF-Soy ___ _________________________________________________________ Consensus BASF_________________________________________________________ ___ 525 ... .I....I....1....1....I....1....1....1....1....1....1....1 AT1 ___ _________________________________________________________ BASF q24 _ _________________________________________________________ AT2 ___ 424 BASF

_ _________________________________________________________ BASF-Corn ___ q29 BASF-Soy ___ _______-_________________________________________________ q00 Consensus BASF_________________________________________________________ ___ 525 ... .I....I....I....I....I....1....1....1....1....1....1....1 BASF_AT1 ___ _________________________________________________________ q24 AT2 ___ _________________________________________________________ BASF q24 _ _____________-___________________________________________ HASF-Corn ___ q29 BASF-Soy ___ _________________________________________________________ q00 Consensus BASF_________________________________________________________ ___ 525 ... .1....1....I....1....1....1....1....1....1....1....1....1 AT1 ___ ______________________--_________________________________ BASF q24 _ _________________________________________________________ AT2 ___ q24 BASF

_ _________________________________________________________ BASF-Corn ___ q29 BASF-Soy ___ _________________________________________________________ q00 Consensus BASF_________________________________________________________ ___ 525 Table 21. ClustalW Analysis of Generic Nucleic Acids 1) afcl (SEQ ID N0:29) 2) AT4g01320 (SEQ ID N0:31 ) 3) AFOO~2ss (SEQ ID N0:33) 4) consensus (SEQ ID N0:70) ....I.:..I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 60 ....I....1....1....1....1....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 120 ....I....I....1....I....1....1....1....1....1....1....1....1 afci ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 180 ....1....I....1....1....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 240 ....I....1....I....I....I....I....I....I....I....I....I....i afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 300 ....I....I....I....i....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 360 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 420 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 480 ....I....I....I....I....1....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 590 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 600 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ 1 AT4g01320 ____________________________________________________________ 1 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 660 ....1....1....1....1....1....1.. .1.. ..
afcl .1.. .1 .I 25 .1..
.I..
________________________________ AT4g01320_________________________________.

TCT

ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX X XX ~CX
~7C~CXX XXXXX 720 afcl 85 AT4g01320 85 Consensus 780 .I ....I.. ..I....I. ...1....1. ...1....1.. ..1..
..1....1....1 afcl a 145 AT4g01320 ~ 145 I

AF007269 ~ 840 Consensus 840 .I ....I....I....I. ...I....1....1....1....1....1....1....1 afcl _____________________________________ AT4g01320 . G_____________________________________ Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....I....1....1....1....1....1....1....1....1..
..1....1....1 afcl __________________________________________TC

G -AT4g01320________________________ I~ 203 AF007269TTTTAGTTTTTTATAATTGCCAGG ~ 960 ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

.I....I....I....I....i....1....1....1....1....1....1....1 afcl ~ 242 AT ~ ~
g Consensus 1020 ...
.I. ...I. ...I.. ..I....I....1....1....1....1....1....1....1 afcl _____________________________________ AT4g01320 _____________________________________ Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1....1....1....1....1....1....1....1....1..
1..
afci .1.. .1 276 . ~
_______________________________________________..

~

AT4g01320_______________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

.I....I....I....I....I....1....1....1....1....1....1....1 afcl e~ ~ i v 336 a :

AT4g01320 ~ ~ 357 ~ I ~ I

AF007269 ~ v 1200 Consensus 1200 .I ....I. ...I.. ..I.... I....1....1....1....1....1....1....1 afci ___________________________________ AT4g01320 ___________________________________ Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....I....I....I....I....I....1....1....1....1....1....1....1 afci ____________________________________________________________ AT4g01320____________________________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

afcl 920 AT4g01320 941 Consensus 1380 ...
.I....1....1....I....1....1....1 ....1....1....1....1....1 fcl . _________________________ ._____________________________ 426 AT4g01320____________________________ _________________________ ACTTACGTTTTTTTATCAGAGATGT

ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXX

....1....1....1....1....1....1.. .. .1.. .1...I..
afci .1 .I.. .1 _______________________________ 454 AT4g01320_______________________________ 975 ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1500 .I..
.I..
.I..
.I..
.I..
.I..
.I..
.1..
.I..
.I..
.I..
.I

afcl ~ 514 AT4g01320 535 Consensus 1560 .I....I....1....I....1....1....1....1....1....1....1....1 ...
afcl . . . _________________________________________________ 525 AT4g01320 ________________________________________________ 546 AF007269 ~ GTTTGATGATTCTGGATTCATCTTATTTCTGAGTTTTTCACATGGATGA 1620 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1620 afci ____________________________________________________________ 525 AT4g01320 ____________________________________________________________ 546 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1680 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ___________________________________ 549 AT4g01320 ___________________________________ 570 AF007269 TGCTTCTCTGAGCATGAAGTTTCTATCTTTTTCCA ~ 1790 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX~~ 1790 ... .I....I....i....l....I....1....1....1....1....1....1....

afci 609 AT4g01320 630 Consensus 1800 .I....I....I....i....I....1....1....1....1....1....1....1 afcl ___________________--________ 639 AT4g01320 ----------------------------- 660 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXX 1860 ....I....I....I....i....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ AT4g01320____________________________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1..
.I..
.I..
.I..
.1..
.1..
.I..
.1..
.I..
.1..
.I..
.I

afcl --- ~ 695 AT4g01320--- i 716 ~

AF007269ATA t 1980 ConsensusXXXX 1980 afcl 751 AT4g01320 772 Consensus 2040 ....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ AT4g01320____________________________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1....1....1...I.. .. I.. .I.. .I..
afcl _______________.I.. . .I.. .I.. .I 795 .I i AT4g01320--------------- ~ 816 AF007269TGTTACATCATACAG ~ 2160 ConsensusXXXXXXXXXXXXXXXX 2160 .1.. .1.. .I.. .I....I....1....1....1....1....1....1....1 afcl _________________________________________ 813 AT4g01320 _________________________________________ 834 AE007269 ' GTACTGTGACTCTTGATGCTTCAAACGAGCTATACTCACATT 2220 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 2220 ....I....I....I....1....1....1....1....1....1..

afci .I..
.1..
.I
___________________________________________ AT4g01320___________________________________________ ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....i....I....I....I....I....1....1....1....1....1....1....1 afcl 889 AT4g01320 910 Consensus 2390 .I.. .I.. .I.. .I....I....I....I....I....I....i....l....l afcl ~... . - ' ..___________________________________________ 906 AT4g01320 __________________________________________ 927 AF007269 ~ GTGAGGCTCAACCGACAGTTCAAAAACTTACTCACATCTACAT 2900 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX 2900 ....I....I....i....i....1....1....1....1....1....1.. .I.. .I
afcl ___________________________________________________.~ ~ 915 AT4g01320 __________________________________________________ 936 AF007269 TTCACTTAAGAAATCATGTCTTATGACCCTCTCTCAATGTTTTGCTTGCA ~ 2960 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX~ 2460 .I....I....I....I....I....1....1....1....1....1....1....1 afcl 975 AT4g01320 996 Consensus 2520 .I.. .1....1....1....1 .I..
.I..
.I..
.1....I..
.I....1..
~

afci r. I : ______________ AT4g01320 I ~ ---------------Consensus XXXXXXXXXXXXXX

....I....I....I....I....I....1....1....1...
.1....1....1....1 afcl ___________________________________________ _________________ AT4g01320___________________________________________ _________________ AAAACTCTAAACCTTTG

ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

afcl ~

AT4g01320 ~

AF007269 ., ~~.
, .~

I
~

ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXX ~
' '~
~~' .1.. ..1....1.. ..1....I... .1....1....1....1....1....1....1 afcl -------------------AT4g01320 ~ --------------------AF007269 ~ TACCATCTTACAATCCCTCA

Consensus XXXXXXXXXXXXXXXXXXX

....I....I....I....I....I....1....1....1....1....1....1....1 afcl ____________________________________________________________ AT4g01320________________________________________________________-___ ConaenausXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....1....I.. .I.. . .I.. .1 .I. .I..
.I..
.I..
.1..
.1..
.1..
.

afcl ----------- C 1141 I~~
~
~

AT4 - ~ 1162 01320 -- ' g --------AF007269TCCTTTTGCAG li~ 2880 ConsensusXXXXXXXXXXXX 2880 ....I.. ..I....I....I....i....1....1....1....1....1....1....1 afcl _________________________________________ AT4g01320 ~

AF007269 e Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....I....I....1....1....1....1....1....1..
.I.. .I
afcl .I.. 1177 .I..
_________________________________________ , AT4g01320 i 1282 I
~

s ConsensusXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

....I....I....I....I....I....1....1....1....1....1....1....1 afcl 1237 AT4g01320 1342 Consensus 3060 ....I....1....1....1....1....1....1....
GTTATATCTCCTGTCTGATTATCACA
~i ....I....1....1....1....1....1....1...

afcl 1275 AT4g01320 1380 Conaenaus 3098 Table 22. ClustalW Analysis of Generic Amino Acids 1) afcl (SEQ ID N0:30) 2) ATago132o (SEQ ID N0:32) 3) Aaoo72s9 (SEQ ID N0:34) 4) consensus (SEQ ID N0:71 ) afcl 58 AT4g01320 60 Consensus Publi 60 ....I....I....I....I....1....1....1....1....1....1....1....1 afci F(----- ~ ~ 113 AT4g01320 ENFNIC ~' ~ ~ 120 AF007269 _______________________________________________________T____ q2 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX~XXXXXXXXXXXXXXXXXXXXX

Publi afcl 173 AT4g01320 180 Consensus 180 Publi afcl 233 AT4g01320 290 Consensus 290 Publi afcl 293 AT4g01320 300 Consensus XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

Publi afcl 353 AT4g01320 360 Consensus 360 Publi ....I....I....I....I....I....1....1....1....1....1....1....1 afci vv v __________________________386 AT4g01320 I~ 1~ ~ ~ TSICVTHLNGFFVGIL 420 AF007269 ~ ~ ~ ~ _______-_________ 278 Consensus XXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGXX920 Publi .I....i....I....I....I....1....1....1....1....1....1....1 afcl ~ H ~ ~ ____________________ q24 AT4g01320 ~ ~ ~ ____________________ 459 AF007269 ~ ~ ~ ____________________ 316 Consensus XXXX ~ ~ ~ -------------------- 980 Publi Table 23. ClustalW Analysis of PPI Nucleic Acids 1) PPI-AtCPP (SEQ ID NO:1) 2) PPI-BnCPP (SEQ ID N0:14) 3) PPI-SoyCPP (SEQ ID N0:17) 4) Consensus (SEQ ID N0:72) PPI-AtCPP 60 PPI-BnCPP 60 PPI-SoyCPP 60 Consensus 60 PPI-AtCPP 120 PPI-BnCPP 120 PPI-SoyCPP 120 Consensus 120 PPI-AtCPP 180 PPI-BnCPP 180 PPI-SoyCPP 180 Consensus ~CX~X~C~CX~X~ 180 PPI-AtCPP 240 PPI-BnCPP 290 PPI-SoyCPP 290 Consensus 240 PPI-AtCPP 300 PPI-BnCPP 300 PPI-SoyCPP 300 Consensus 300 PPI-AtCPP 360 PPI-BnCPP 360 PPI-SoyCPP 360 Consensus 360 .1.. .1.. .I.. .I.. .I.. .I.. .1.. .1.. .I.. .I.. .I.. .1 PPI-AtCPP ~~ ~ 420 PPI-HnCPP 420 PPI-SoyCPP C C T ~ T ~ C T ~ T T 420 Consensus 420 PPI-AtCPP 980 PPI-BnCPP 480 PPI-SoyCPP 980 Consensus 980 ....I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP 590 PPI-BnCPP 590 PPI-SoyCPP 540 Consensus 590 ....I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP 600 PPI-BnCPP 600 PPI-SoyCPP 600 Consensus 600 .I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP 660 PPI-BnCPP 660 PPI-SoyCPP 660 Consensus 660 .I....I....I....I....1....1....1....1....1....1....1....1 PPI-AtCPP 720 PPI-BnCPP 720 PPI-SoyCPP 720 Consensus 720 .I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP 780 PPI-BnCPP 780 PPI-SoyCPP 780 Consensus 780 .I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP T 890 PPI-BnCPP ~h~ e~ C 890 PPI-SoyCPP C C ~~ C T T T 890 T
C
T T TT T~C GGT T T ~ CC T T
X X XX X
C G t t T T ~ T T T ~ ~ T
C T ~~T~~ ~ ~ TC
X
r.
.

T . I~
C
I

C C C ~
C

X

T.. r..
.~ T..
.

C~ ~T C T G
C

Consensus ~C~~C~C~C~CX~1C~CX~C~C~X

PPI-AtCPP 900 PPI-BnCPP 900 PPI-SoyCPP 900 Consensus 900 PPI-AtCPP 960 PPI-BnCPP 960 PPI-SoyCPP 960 Consensus 960 PPI-AtCPP 1020 PPI-BnCPP 1020 PPI-SoyCPP 1020 Consensus 1020 PPI-AtCPP 1080 PPI-BnCPP 1080 PPI-SoyCPP 1080 Consensus 1080 PPI-AtCPP 1140 PPI-BnCPP 1140 PPI-SoyCPP 1190 Consensus 1140 PPI-AtCPP 1200 PPI-BnCPP 1200 PPI-SoyCPP 1200 Consensus 1200 .I....I... .I.... I. ...I. ...1....1.... 1... .1....1... .1....1 PPI-AtCPP ~ C 1260 PPI-BnCPP ~ C 1260 PPI-SoyCPP T T C T GC CC
GGCC G T

Consensus X XXX XX

....1....1....1 PPI-AtCPP1275 PPI-BnCPP1275 PPI-SoyCPPG C 1275 ConsensusX 1275 Table 24. ClustalW Analysis of PPI Amino Acids 1) PPI-AtCPP (SEQ ID N0:2) 2) PPI-BnCPP (SEQ ID NO:15) 3) PPI-SoyCPP (SEQ ID NO:1H) 4) consensus (SEQ ID N0:73) PPI-AtCPP 60 PPI-BnCPP 60 PPI-SoyCPP 60 Consensus PPI 60 ....1....1....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP 120 PPI-BnCPP 120 PPI-SoyCPP 120 Consensus 120 PPI

PPI-AtCPP 180 PPI-BnCPP 180 PPI-SoyCPP 180 Consensus 180 PPI

PPI-AtCPP 290 PPI-BnCPP 290 PPI-SoyCPP 290 Consensus 240 PPI

. I.. .. ..1.. .I...1....l.. I..
PPI-AtCPP~ .I.. .I.. .I.. ~ .I.. .I
.I ' I II ~ . 300 . ~
~' PPI-BnCPP~ I II'L QN_EN T S
~ ~ I 300 PPI-SoyCPP~ II~ I ~ ~~ 300 ~

Consensus~ ~ XXX 300 PPI

. I....I....I....I....1....1....1....1....1....1....1....1 PPI-AtCPP.~ ~ ~ ~ ' ~~ 360 PPI-BnCPPV~ t T~ ~ ~ D ' 360 ~

PPI-SoyCPP ~ v v 1 360 ConsensusX~ X ~ ~ W ~ ~ 360 PPI ~

PPI-AtCPP 420 PPI-BnCPP 420 PPI-SoyCPP 420 Consensus PPI 420 .I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP ~.~ -_______________________________________________________ 424 PPI-BnCPP T ________________________________________________________ q24 PPI-SoyCPP ________________________________________________________ 424 Consensus PPI 480 Table XX. ClustalW Analysis of PPI/Generic Nucleic Acids I) PPI-AtCPP (SEQ ID NO:I) 2) PPI-BnCPP (SEQ ID N0:14) 3) PPI-SoyCPP (SEQ ID N0:17) 4) afci (SEQ ID N0:29) S) Axagoi32o (SEQ ID N0:31) 6) AFOO~2ss (SEQ ID N0:33) 6) Consensus (SEQ ID N0:75) PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP
______________________________________________________________________ afcl _____________-________________________________________________________ AT9g01320 ______________________________________________________________________ ATGGCGATTCCTTTCATGGAAACCGTCGTGGGTAAGCTTCAAAACCTTTTTCTGAGACATTTTACTATCC

Consensus ______________________________________________________________________ PPI-AtCPP ....
NA ....
....
....1....1....
....1....1....1....I....1....1....1....
----------ATGGCGTTTCCCTACATGGAAGCCGTTGTCGGATTTATGATATTAATGTACATTTTTGAA

PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP----------ATGGCGTTTCCCTACATGGAAGCCGTTGTCGGATTTATGATATTAATGTACATTTTTGAA

afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ TGTTTCACTCATCGTATTTCGTTTTTGTTTGGGTTTTGCTTTCTGTGTTGTGTGTGTTGAGATTCCATGA

Consensus ----------ATGGCGATTCCTTTCATGGAAACCGTCGT-GGTTTTATGATAT--ATGTACATTTTTGAA

PPI-AtCPP
....1....I....I....1....I....1....1....1....1....1....1....1....1....
NA ACTTACTTGGATG-TGCGACAACATAGGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAGGGTGTT

PPI-BnCPP
______________________________________________________________________ PPI-SOyCPPACTTACTTGGATG-TGCGACAACATAGGGCCCTCAAACTTCCTACTCTTCCAAAGACTTTAGAGGGTGTT

afcl ______________________________________________________________________ AT9g01320 ______________________________________________________________________ CTCGTTTGTTTCATATACCATCGTCTCTGCTTCTCGTTTCTAAATTTTGTTCTTTTCTAATAGTGCGTA

Consensus --CTATTTGGAT----TGGCAACATG----CCTCAA--CTTCCACTCTCC---AAACTTGGTGGTGTAT-....I....1....I....1....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP
ATCAGCCAAGAGAAATTTGAGAAATCTAGAGCCTATAGTCTTGATAAAAGCCACTTCCATTTTGTTCACG
NA

PPI-BnCPP
______________________________________________________________________ PPI-SoyCPPATCAGCCAAGAGAAATTTGAGAAATCTAGAGCCTATAGTCTTGATAAAAGCCACTTCCATTTTGTTCACG

afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ GATTTGATTTGAG

Consensus ---AGCCAAGAGAAGTTTGAGAAATCTGAG--CTACAGTCTTGAAAAAG--CATT--CATTT-GTTCA-G

PPI-AtCPP NA AGTTTGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGGGGTATTGCCCTGGTTTTGGAA-----G
PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP AGTTTGTGACAATAGTGACAGACTCTACAATTTTGTACTTTGGGGTATTGCCCTGGTTTTGGAA-----G
afcl ______________________________________________________________________ AT9g01320 ______________________________________________________________________ TTTCATCATCTCTTCCTCGGTTTTAGAATGTACGGAGCTTCTCTGTTAACCAAAATCTAG
Consensus AGTTTGTA--CATAGT--TAGACTCT-CAATTTTGT-CTTTGGG---TTTGCCTGGTTTTGGAA-----G

....1....1....1....I....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA
AAATCAGGAGATTTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCATACCCTTGCCTTCT
PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP
AAATCAGGAGATTTTATGACAATAGCTGGTTTCAATGCTGAGAATGAAATACTGCATACCCTTGCCTTCT
afcl ______________________________________________________________________ AT9g01320 ______________________________________________________________________ GATTTGGGAAGAAAAGTCGGAGTCTTTTTTTTCCTCATTCCCGATTGGAAATTGAGAATCTTGAAATTTT
Consensus AT-TCGGG---TTTTGCAA------TTGGT-----CATCGAGAATGAAAT-CTGCATACC-TT--CTTCT

....I....1....I....1....I....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA TAGCAGGGCTGATGATTTGGTCACAGATAACAGATTTGCCCTTTTCTCTGTACTCAACTTTT-----GTG
PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP TAGCAGGGCTGATGATTTGGTCACAGATAACAGATTTGCCCTTTTCTCTGTACTCAACTTTT-----GTG
afcl ______________________________________________________________________ AT9g01320 ______________________________________________________________________ TCTTTGTTCAAGTCATACkIGCTTGAGGTTTTGGGTTTTCTTGTCAGGGTATTATTATGTTCGTGACTGCA
Consensus T-GCGGT----ATGAT--GGTCACAGATA--CGATTTGCCTTTTCTT--GTACTCAACTTT------GTG

....I....1....1....I....1....I....I....1....1....1....1....1....1....1 PPI-AtCPP NA ATTGAGGCCCGTCATGGTTTTAATAAGCAAACAC--CATGGTTATTCTTTAGGGACATGCTTAAAGGAAT
PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP ATTGAGGCCCGTCATGGTTTTAATAAGCAAACAC--CATGGTTATTCTTTAGGGACATGCTTAAAGGAAT
afcl ______________________________________________________________________ AT9g01320 _____-_________________________________________________________________ ACTAGAGTTTTCTGGAGTTTTTTGAAATGGGTTTTGTGTTGTGGAACCGTATGTGAATGTTGCATCAAAA
Consensus AT--GAGTCCG-CATGGTTAAAACAAACA-------CATGGTT---TCTTAGGGACATG--TAAAGGAAT

....I....I....I....1....1....1....I....1....1....1....1....1....1....1 PPI-AtCPP NA TTTCCTTTCTGTAATAATTGGTCCACCTATTGTGGCTGCAA------TCATTGTAA-TAGTACAGAAAGG
PPI-BnCPP ___________________________________________-__________________________ PPI-SoyCPP TTTCCTTTCTGTAATAATTGGTCCACCTATTGTGGCTGCAA------TCATTGTAA-TAGTACAGAAAGG
afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ CTCTTTCAGTGCTCCAATGTTTCCATCAGTAGTCAGCACAAGAGATCTTTTTATATCTGGTTGATCAAAA
Consensus TTCCTTCTGTATA------G--CC-CCTATTGTG-CTGCAA------T-ATTGTA--TAGT-CAGAAAGG

PPI-AtCPP NA AGGTCCATA--CTTGGCCATCTATCTTTGGGTTTTTACGTTTGGTCTTTCTATTGTGATGATGACCCTT
PPI-BnCPP ____________________________________________________________________ PPI-SoyCPP AGGTCCATA--CTTGGCCATCTATCTTTGGGTTTTTACGTTTGGTCTTTCTATTGTGATGATGACCCTT
afcl ____________________________________________________________________ AT4g01320 ____________________________________________________________________ AAGTAGATGATGTTATTGAATTTTCAGTGATGGAGTATCTGTTGTTGTGGCATTTAGAGTAGATTCGTA~
Consensus AGGTCC-----TATG-CCATCTATCTTGGG---------TTTAGTTTTCTTCTTGTGATGATGACC
,~~,T

.I....I....I....i....l....l....1....1....1....1....1....1....1 PPI-AtCPP NA AT C GCTCC CT T TAAG C CTCC C -- CC G C CT GGGAGAA
PPI-BnCPP G T C --T C~GG C G GTTGGT T~TGAT G GT C TT T G GTATTTGG
PPI-SoyCPP AT C GCTCC CT T TAAG C CTCC~C -- CC ~ G C CT GGGAGAA
afcl G T C~--T C~ GG C GTGGGT T TGAT G GT C~ TT T G GTATTTGG
AT4g01320 G T C --T C~ GG C GTGGGT T TGAT G GT C TT T G GTATTTGG
AF007269 TT TC T GTT ~ TCTTTTTC TACAGGT T GAT G GT C~ TT G GTATTTGG
Consensus A- C-------- GCCCCT(aT -AA C~CTCC-C -- CC GG--- CT -GGGAGAA
PPI-AtCPP NA
PPI-BnCPP
PPI-SoyCPP
afcl AT4g01320 Consensus ..I....I....I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPPTC CACC GC TAT T TTCTTCAAGAACAAGAGGATTGTCC---CTTATGACAC
NA T

PPI-BnCPP-A TT CT -GCTC ______________________________________ PPI-SoyCPPTC CAC C GC TAT T TTCTTCAAGAACAAGAGGATTGTCC---CTTATGACAC
T

afcl -A TT C ._GCC ______________________________________ AT4g01320-A TT C _GC C _______________________________________ AF007269-A TT ' C -GC C TCTTGACAAAAGGTTTCGTCTTGATCATATTTATATCAT

Consensus-TC -CAT - GCT--T T --TTCTTAAGAACAA-AGGATTGTC-----TTATGACAC

PPI-AtCPP NA ATTAATTC-------------AACAG~GCA~AGAC-GATGmGGAA~T~G~TG-CT~T~GC~CA~G
PPI-BnCPP_________________________________________TCTTGACAAA--CC TT C TT

PPI-SoyCPPATTAATTC-------------AACAG~GCA~AGAC-GATG~GGAA~T~G~TG-CT
T GC C G

afcl -----------------------------------------TCTTGACAAA--C TT C TT

AT4g01320-----------------------GGTCACTGAGTTT TGC C TT C TT
T

AF007269TTTAGTTTTTTATAATTGCCAGGGG~ C TT C
n~' ,~~,'n'TC~TCACTGAG TT
n~'~~CTTT TGC

ConsensusATTATTC---------------ACAGC ------ G T G---CT GC ---AGA
C C
~

PPI-AtCPP NA
PPI-BnCPP
PPI-SoyCPP
afcl AT4g01320 Consensus .1.. .1....1....I....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA TATACACTAGTGCGAAATTCAGCTGATCTGTATCGAAGCTTTGGGTTTGATACGCAGCCAGT
PPI-BnCPP _______________________________________________________________ PPI-SOyCPP TATACACTAGTGCGAAATTCAGCTGATCTGTATCGAAGCTTTGGGTTTGATACGCAGCCAGT
afcl _______________________________________________________________ AT4g01320 _______________________________________________________________ Consensus TACAC-CTAGTG--AAATCC---TGATCT----TGAG----TTGGTTTGATAC-CAGCCG--....1....1....1....1....1...1....1.. .. ....1....1 PPI-AtCPPCCTCATTGGGCTCATCATATTTCAGC.I.. .1.. T--NA .I .1..
. .1 CTGTA~T~CCAC .
.C TT
.
C
.T

PPI-BnCPP-------------------------TCTGGCGGC-TT CT C -T G C C CAG

PPI-SoyCPPCCTCATTGGGCTCATCATATTTCAGCCTGT TCCACC TT C T -afcl -------------------------GTCTG T-GTT C T G C CAG

AT4g01320-------------------------GTCTG T-GTT C T G C CAG

AF007269TTGTAAAGTTTTCATTTTTACCTTAGTCTG T-GT C = T G C CAG

ConsensusTCTCATTGG---TATCATATTTCAGCCTGT TC-ACC -----CATGT T C ------.I....I....I....I....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA ~~CCT CAGC GAT T G ~ TC GC C GCTT GATATGCATCTGGAT
PPI-BnCPP G CCT C C~ CTT G C-T T GTCAC --------------PPI-SoyCPP CCT CAGC GAT T G ~ TC GC T C GCTT GATATGCATCTGGAT
afcl G CT T T C~ CTT -T T GTCAC --------------AT4g01320 'TG CT T T C~ CTT -T T GTCAC ---------------AF007269 ~ TG ~ CT T T C~ CTT -T T GTCAC GTGTTCCAAATAAAC
Consensus ~~CCTG------T GCG TCA~A~-C TIC--- GCTT G-TATGCAGTCGG--PPI-AtCPP NA TACGCGGTG--GTCTTGTGAAACTACAGGAGGAGAATCTGTCAGCT----ATGAATACAGATCCTTGGTA
PPI-BnCPP
______________________________________________________________________ PPI-SOyCPP TACGCGGTG--GTCTTGTGAAACTACAGGAGGAGAATCTGTCAGCT----ATGAATACAGATCCTTGGTA
afcl __________-___________________________________________________________ AT4g01320 ______________________________________________________________________ CCCTTCATATAGTCCTATACGTTTAGCATCAAAATATCTATTTTCTTAAGATAATAATATTTCTTTTATA
Consensus --T--------GTCTAGTGAA-CTACAGGAGAGAA---TGTCAGC-----ATGAA-ACAGATCCTTG-TA

....I....I....1....1....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA CTCT---GCTT T C CATCC CC C ----- GT G GCCGCGC G ---i PPI-BnCPP ---------- G T G~CAT TG-- C CTT C ------ T G----PPI-SoyCPP CTCT---GCTT~ T C CATCC CC C ----- GT G G T GCCGCGC G ---afcl ---------- G T CAT TG-- C~ CTT C ----- T G----AT4g01320 ---------- G T CAT TG-- C CTT C -----==T G----AF007269 TTCTGATGCA G T CAT TG--- C~ CTT C ----- T G----Consensus CTC----GCTT~ TCCAC TCCC G GAAAG GAG GAGATAATCT TTCT

.. .1....1....1....1....1....1....1....1....1....1....1....1....1....1 PPI-AtCPP NA --C GGAT G GAA T~~g______________________________________________ PPI-BnCPP --T CG TTC~ ______________________________________-_______ PPI-SoyCPP --C GGAT G GAA T~ ______________________________________________ afcl --T CG TTC ___________________________________________-__ AT4g01320 --T CG TTC~~ .. _______________________________________________ AF007269 --T CG TTC ~ GTATGTCGTATTTCCAACACTACCTTGTGACTTACGTTTTTTTATCA
Consensus TTC TTT ~ GGT ~ ~ GTATGTCGTATTTCCAACACTACCTTGTGACTTACGTTTTTTTATCA

PPI-AtCPP NA _________________-_-_______--_________________________________________ PPI-BriCPP ____________-__-____---________________~,~w,»vwvrNervrerWrwWV~e~aer_«vraa PPI-SoyCPP __________________________________________________________-___________ afcl ___________________________-__________ AT4g01320 _____________________________-________ .~.. ~.- ., Consensus GAGATGTGGATTAAATTTGCTTCTAAATTCTGTTGACAGt~7~~U)ayyyyecelyyeYWV~)_~WV~e4dgW
lewela PPI-AtCPP NA _________________________________-__________________________________-_ PPI-BnCPP T~C CT~C~TmTmC~CmA~T~G~T~'f~--PPI-SoyCPP ________________-_____________________________________________________ afcl AT4g01320 Consensus PPI-AtCPP NA _________--___________________________________________________________ PPI-BnCPP __________________________-________________________-_______-__________ PPI-SOyCPP ____________-__________-________________________-__-__________________ afcl _____________-_______________________-___________________________-____ AT9g01320 ________-___-___________________-_____________________________________ TTGATGATTCTGGATTCATCTTATTTCTGAGTTTTTCACATGGATGACTATTCTCCATTGAGTGTGAGCT

Consensus TTGATGATTCTGGATTCATCTTATTTCTGAGTTTTTCACATGGATGACTATTCTCCATTGAGTGTGAGCT

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP ___________-__________________________________________________________ afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ TCAAAGTTTTTAGTTTTCGTGTTAAAAATTTAAAATTTGCTTCTCTGAGCATGAAGTTTCTATCTTTTTC
Consensus TCAAAGTTTTTAGTTTTCGTGTTAAAAATTTAAAATTTGCTTCTCTGAGCATGAAGTTTCTATCTTTTTC

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP -- CmC
PPI-SoyCPP ______________________________________________-_______________________ afcl AT4g01320 Consensus PPI-AtCPP NA _____-________________________________________________________________ PPI-BnCPP ~TmT~T~TmT ______________________ PPI-SoyCPP
______________________________________________________________________ afcl C C ______________________ AT4g01320 C C ----------------------Consensus C C GTGTGTATTTCTGTCATGGCCAT

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP
______________________________________________________________________ PPI-SOyCPP
______________________________________________________________________ afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ TTTACAATTCACTGCTTGTTTGCATATGTTGTTACCAGACAATATAATCTCCCGCTTTTTTATGGCTATA
Consensus TTTACAATTCACTGCTTGTTTGCATATGTTGTTACCAGACAATATAATCTCCCGCTTTTTTATGGCTATA

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP -~T C
PPI-SoyCPP
______________________________________________________________________ afcl AT4g01320 Consensus PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ~___________________________ PPI-SoyCPP
______________________________________________________________________ afcl . C.. __________________________ AT9g01320 . C.. __________________________ AF007269 ' C~ TGAGAAGCTTGAGATCTCTTCCTACCT
Consensus ' C~~ TGAGAAGCTTGAGATCTCTTCCTACCT

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP _____________________________________________________ PPI-SoyCPP ________________-_____________________________________________________ afcl AT4g01320 _____________________________________________________ . .

Consensus ACTTTACTCTAGTTTACCATTAGAAGCTTACGTATCTTGTTACATCATACAG ~li ~~~

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ~C~~ C~A~-________________________ PPI-SoyCPP
______________________________________________________________________ afcl AT4g01320 Consensus PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP _____________________________________________________________~C
PPI-SoyCPP
______________________________________________________________________ afcl _____________________________________________________________ . ..
AT4g01320 _____________________________________________________________ . ..
AF007269 CGAGCTATACTCACATTTCTGTTTCTGGTTCTGAAACATAACATAATCTTCTATTGTGCA ~ ~' Consensus CGAGCTATACTCACATTTCTGTTTCTGGTTCTGAAACATAACATAATCTTCTATTGTGC ~A

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP G~C
PPI-SoyCPP ______________________-_______________________________________________ afcl AT9g01320 Consensus PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ~T~________________________________________________________ PPI-SoyCPP
______________________________________________________________________ afcl ________________________________________________________ AT4g01320 ~ . ________________________________________________________ AF007269 ' GTGAGGCTCAACCGACAGTTCAAAAACTTACTCACATCTACATTTCACTTAAGAAA
Consensus ~~GTGAGGCTCAACCGACAGTTCAAAAACTTACTCACATCTACATTTCACTTAAGAAA

PPI-AtCPP NA _-____________________________________________________________________ PPI-BnCPP _____________________________________ v r_~r~1y~11~re1eW
ele7yy~ye~fi PPI-SoyCPP
______________________________________________________________________ afcl _____________________________________ AT9g01320 _____________________________________ AF007269 TCATGTCTTATGACCCTCTCTCAATGTTTTGCTTGCA ~ ~~ ~~ ~ ~~ ~
Consensus TCATGTCTTATGACCCTCTCTCAATGTTTTGCTTGCA

PPI-AtCPP NA _____________________________________-________________________________ PPI-BnCPP
PPI-SoyCPP ________________________________________________________________-_____ afcl AT9g01320 Consensus PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ~__________________________________________________________ PPI-SoyCPP
______________________________________________________________________ afcl ~ ~ ~- - , -_________________________________________________________ AT4g01320 ~ . . . __________________________________________________________ AF007269 ~ GTTTGTTATTTTTGCCTTTTGACACTAATCTAATGAATCAAGGATGGATTAAGAAAAA
Consensus ~ GTTTGTTATTTTTGCCTTTTGACACTAATCTAATGAATCAAGGATGGATTAAGAAAAA

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ___________________________________________ T~Cm PPI-SoyCPP
______________________________________________________________________ afcl ___________________________________________ T
AT4g01320 ___________________________________________ .T
AF007269 AAAACTCTAAACCTTTGGTTATATCTCCTGTCTGATTATCACA ~~~~~ ~m ~ T
Consensus AAAACTCTAAACCTTTGGTTATATCTCCTGTCTGATTATCACA ~~~~ I~ ~i~~ G ~~~ ~T~

....I....I....I....I....I....I....I....1....1....1....1....1....1....1 ....I....I....I....I....I....I....I....1....1....1....1....1....1....1 PPI-AtCPP NA _-____________________________________________________________________ PPI-BnCPP C~'i' ______________________ PPI-SoyCPP _______________________________________________________________-______ afcl C - ______________________ AT4g01320 C ~ ~~. _______________________ AF007269 C ~la~ TACCATCTTACAATCCCTCAAGA
Consensus C ~e ~~ TACCATCTTACAATCCCTCAAGA

PPI-AtCPP NA ________________________________________________________________-_____ PPI-BnCPP ______________________________-____________________________________ PPI-SoyCPP
______________________________________________________________________ afcl ____________________________________________________________________-_____________________________________________________________________ Consensus TCCAACCATAGTTTCTTTATTGCAATGGCAGCCTCATCTACTAATCTGAGTTAACGTTCCTTTTGCAG

PPI-AtCPP NA _________________________________-____________________________________ PPI-BnCPP ~~f~ C~G~-____ PPI-SoyCPP ___________________________________-__________________________________ afcl AT4g01320 Consensus PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP
______________________________________________________________________ afcl ________________________________________________-_____________________ AT4g01320 GAAGATAACAACAGAACACAAACTGTTACCTCAATTTGTGTCACACACTTAAATGGATTTTTTGTTGGGA

GAAGATAACAACAGAACACAAACTGTTACCTCAATTTGTGTCACACACTTAAATGGATTTTTTGTTGGGA
Consensus GAAGATAACAACAGAACACAAACTGTTACCTCAATTTGTGTCACACACTTAAATGGATTTTTTGTTGGGA

PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP ______~__ GC~T Cm PPI-SoyCPP _______________________________________________________________-______ afcl AT4g01320 Consensus PPI-AtCPP NA ________________________________________________________ PPI-BnCPP
PPI-SoyCPP ________________________________________________________ afcl T
AT4g01320 T ~~~ ~ R
AF007269 T~' a~ ~ I ~
Consensus T v ~~~ ~v~~
Table XX. ClustalW Analysis of PPI/Generic Nucleic Acids 1 ) PPI-AtCPP (SEQ ID NO:1 ) 2) PPI-BnCPP (SEQ ID N0:14) 3) PPI-SoyCPP (SEQ ID N0:17) 4) afcl (SEQ ID N0:29) 5) AT4g01320 (SEQ ID N0:31) 6) AFOOn69 (SEQ ID N0:33) 6) Consensus (SEQ ID N0:75) PPI-AtCPP NA
______________________________________________________________________ PPI-BnCPP
______________________________________________________________________ PPI-SoyCPP ______________________________-_______________________________________ afcl ______________________________________________________________________ AT4g01320 ______________________________________________________________________ ATGGCGATTCCTTTCATGGAAACCGTCGTGGGTAAGCTTCAAAACCTTTTTCTGAGACATTTTACTATCC

Table 26. ClustalW Analysis of PPI/Generic Amino Acids 1) PPI-AtCPP(SEQ ID N0:2) 2) PPI-BnCPP(SEQ ID NO:IS) 3) PPI-SoyCPP(SEQ ID N0:18) 4) afcl (SEQ ID N0:30) 5) AT4g01320(SEQ ID N0:32) 6) AFOO72s9 (SEQ ID N0:34) 7) Consensus(SEQ ID NO:74) Gener PPI-AtCPP 58 PPI-BnCPP 58 PPI-SOyCPP 58 afcl 58 AT4g01320 60 Consensus Gener 60 .I.. .I.. .I.. .I.. .~.. .~.. .~.. .~....~.. .I.. .I..
y ~n n PPI-AtCPP --- ~ ~T ~ T ~ ~ . DFT~ITI FPI ~ ~ 113 PPI-BnCPP ----L~' ~ ~ ~ I GFeLPM LDP ~ 113 PPI-SoyCPP ----Fi T~ T ~ ~ K DF'N~TI F'N ~ 113 afcl -----Y ~ ~ '.~, PRL LDP S~ 113 AT4g01320 ENFNICSY ~ ~ T VIrPRL LIP S 120 AF007269 _____________________________-______________________________ 41 Consensus Ganer XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

PPI-AtCPP 1/S
PPI-BnCPP 173 PPI-SoyCPP 173 afci 173 AT4g01320 180 Consensus Gener 180 PPI-AtCPP 233 PPI-BnCPP 233 PPI-SoyCPP 233 afcl 233 AT4g01320 240 Consensus Gener 290 PPI-AtCPP 293 PPI-BnCPP 293 PPI-SoyCPP 293 afcl 293 AT4g01320 300 Consensus Gener 300 PPI-AtCPP 353 PPI-BnCPP 353 PPI-SoyCPP 353 afcl 353 AT4g01320 360 Consensus Gener 360 .1.. .1.. ...1.. .1..
PPI-AtCPP S .1.. g GG .1....1....1....1....1....1 .1 __________________________ i, R

PPI-BnCPP II~ ~ ____-__-__________________ 386 ~
I

PPI-SoyCPP S S G --____-___________________ 386 n afcl ~ ~ ____-_____________________ 386 AT4g01320 ~ ~ REDNNRTQTVTSICVTHLNGFFVGIL 920 AF007269 ~ ~ REDNNRTQT----------------- 278 Consensus ~ X XXXXXXXXXXXXXXXXXXXXXXXXXX 420 Gener X
X

.I....1....1....1....1....1. ...1....
PPI-AtCPP ~ ~_ EP AE 924 PPI-BnCPP ~ ~ ~929 PPI-SoyCPP ~ L ~EP~E 429 w afcl ~ ~ ~429 AT4g01320 ~ ~ 459 AF007269 ~ ~ ~316 Consensus X ~ X X 459 Gener

Claims (45)

CLAIMS:

1. A method of producing a transgenic plant, comprising introducing into a plant cell a compound that increases prenyl protease expression or activity to generate a transgenic cell; and regenerating a transgenic plant from said transgenic cell.

2. The method of claim 1, wherein said plant has an altered phenotype selected from the group consisting of increased tolerance to stress, delayed senescence, increased ABA sensitivity, increased yield, increased productivity and increased biomass compared to a wild type plant.

3. The method of claim 1, wherein said compound comprises a nucleic acid sequence encoding prenyl protease.

4. The method of claim 3, wherein said nucleic acid comprises SEQ ID NO: 1,14, 17, 68, 70, 72, 74, 21, 23, 25, 27, 29, 31, or 33.

5. The method of claim 3, wherein said nucleic acid is operably linked to a promotor.

6. The method of claim 5, wherein said promoter is selected from the group consisting of a constitutive promoter, an ABA inducible promoter, tissue specific promoters or a ward cell-specific promoter.

7. The method of claim 1, wherein said compound is a prenyl protease polypeptide or fragment thereof.

8. The method of claim 7, wherein said prenyl protease polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 15, 18, 22, 24, 26, 28, 30, 32, 34, 69, 71, 73, or 75.

9. The transgenic plant produced by claim 1.

10. The seed produced by the transgenic plant of claim 9, wherein said seed produces a plant that has an altered phenotype selected from the group consisting of increased tolerance to stress, delayed senescence, increased ABA sensitivity, increased yield, increased productivity and increased biomass compared to a wild type plant.

11. A method of producing a transgenic plant, comprising introducing into a plant cell a nucleic acid that inhibits prenyl protease expression or activity to generate a transgenic cell; and regenerating a transgenic plant from said transgenic cell.

12. The method of claim 11, wherein said plant has an altered phenotype selected from the group consisting of increased tolerance to stress, delayed senescence, increased ABA sensitivity, increased yield, increased productivity and increased biomass compared to a wild type plant.

13. The method of claim 11, wherein said nucleic acid comprises an antisense nucleic acid sequence encoding prenyl protease.

14. The method of claim 13, wherein said antisense nucleic acid comprises 20 or more consecutive nucleic acids complementary to SEQ ID NO: 1, 14, 17, 21, 23, 25, 27, 29, 31, 33, 68, 70, 72, or 74.

15. The method of claim 13, wherein said antisense nucleic acid comprises SEQ
ID
NO: 16,19, 20, 5, 35, 37, 38, 42, 43, 45 , 46, 48, 49a 51, or 52.

16, The method of claim 11, wherein said nucleic acid is operably linked to a promotor.

17. The method of claim 16, wherein said promoter is selected from the group consisting of a constitutive promoter, an ABA inducible promoter, tissue specific promoters or a guard cell-specific promoter.

18. The method of claim 11, wherein the nucleic acid is an inhibitor of farnesylation.

19. The transgenic plant produced by any one of the methods of claims 11.

20. The seed produced by the transgenic plant of claim 19, wherein said seed produces a plant that has an altered phenotype selected from the group consisting of increased tolerance to stress, delayed senescence, increased ABA
sensitivity, increased yield, increased productivity and increased biomass compared to a wild type plant.

21. A method of producing a transgenic plant, comprising introducing into a plant cell a nucleic acid selected from the group consisting of SEQ ID NO: 16, 19, 20, 5, 35, 37, 38, 42, 43, 45, 46, 48, 49, 51, and 52 to generate a transgenic cell; and regenerating a transgenic plant from said transgenic cell.

22. An isolated polypeptide comprising the mature form of an amino acid sequenced selected from the group consisting of SEQ ID NO: 2, 15, 18, 69, 71, 73 and 75.

23. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 18, 69, 71, 73 and 75.

24. An isolated polypeptide comprising an amino acid sequence which is at least 96% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15.

25. An isolated polypeptide comprising an amino acid sequence which is at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, and 18.

26. The polypeptide of claim 24, wherein said polypeptide has prenyl pretease activity.

27. An isolated polypeptide, wherein the polypeptide comprises an amino acid sequence comprising one or more conservative substitutions in the amino acid sequence selected from the group consisting of SEQ ID NO: 15, and 18.

28. The polypeptide of claim 23, wherein said polypeptide is naturally occurring.

29. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 14, 17, 20, 16, 19, 68, 70, 72, and 74.

30. The nucleic acid molecule of claim 29, wherein the nucleic acid molecule is naturally occurring.

31. An isolated nucleic acid molecule encoding the mature form of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO: SEQ ID NO: 2, 15, and 18.

32. An isolated nucleic acid molecule, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 14, 17, 20, 16, 19, 68, 70, 72, and 74.

33. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 94% identical to the nucleotide sequence selected from the group consisting of SEQ ID NO: 17, 18 and 19.

34. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 99% identical to the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 14, 17, 20, 16, and 19.

35. A vector comprising the nucleic acid molecule of claim 29.

36. The vector of claim 35, further comprising a promoter operably linked to said nucleic acid molecule.

37. A cell comprising the vector of claim 36.

38. An antibody that immunospecifically binds to the polypeptide of claim 22.

39. The antibody of claim 38, wherein the antibody is a monoclonal antibody.

40. The antibody of claim 37, wherein the antibody is a polyclonal antibody.

41. A method of identifying an agent that binds to the polypeptide of claim 27, the method comprising:
(a) introducing said polypeptide to said agent; and (b) determining whether said agent binds to said polypeptide.

42. The method of claim 41, wherein the agent is a farnesylation inhibitor.

43. A method for identifying farnesylation modulator, the method comprising:
(a) providing a cell expressing the polypeptide of claim 22;
(b) contacting the cell with a candidate substance; and (c) determining whether the substance alters farnesylation activity;
whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition in the absence of the substance, the substance is identified as a farnesylation modulator.

44. A method for identifying an interacting gene of prenyl protease, the method comprising:
a) providing the transgenic plant of claim 1;
b) creating a library of mutagenized plants from (a);
c) determining whether the mutagenized plant contains an altered phenotype;

whereby, the mutagenized plant has altered the function of an interacting gene of prenyl protease which results in an altered phenotype from the transgenic plant of (a) to that of a wild type non-transgenic plant.

45. A plant; wherein a mutation has been introduced in the gene encoding prenyl protease, resulting in said plant displaying altered prenyl protease activity and an increased tolerance to stress as compared to a wild type plant.